Compositions and methods for delivering an agent to a wound

ABSTRACT

The invention provides compositions featuring chitosan and methods for using such compositions for the local delivery of biologically active agents to an open fracture, complex wound or other site of infection. Advantageously, the degradation and drug elution profiles of the chitosan compositions can be tailored to the needs of particular patients at the point of care (e.g., in a surgical suite, clinic, physician&#39;s office, or other clinical setting).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 U.S. national entry ofInternational Application PCT/US2010/027481 (WO 2010/107794) having anInternational filing date of Mar. 16, 2010 which claims the benefit ofthe following U.S. Provisional Application Ser. Nos. 61/160,539, filedMar. 16, 2009, 61/171,805, filed Apr. 22, 2009, and 61/227,606, filedJul. 22, 2009; the entire contents of each of which are incorporatedherein by this reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the following grants from the U.S. Army:AMEDD Grant No. W81 XWH-080312. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

The skin serves as an important barrier to infection. Any trauma thatbreaks the skin creates an opportunity for pathogen entry and infection.Open fractures are ideal sites for infection. Surgical site infectionsin closed fractures range from 3.6-8.1%. In contrast, surgical siteinfections in open fractures range from 17.5-21.2%. Pathogens present atthe site of an open fracture may create not only local infections, butcan also cause serious infections in the bone and associated tissues.Complex open wounds are also prone to infection with a number ofbacteria. The type of bacteria infecting the wound typically variesdepending on the cause of the trauma. To reduce the risk of infection,the current standard of care involves debridement, irrigation, andsystemic antibiotic therapy. Even with aggressive therapies and systemicantibiotic treatment, infections remain a significant source ofmorbidity and mortality. Tissues compromised by trauma and infectionoften have reduced vascularization, which limits the delivery ofcirculating therapeutics. Increased concentrations of systemicantibiotics are usually required to compensate for poor circulation inthe damaged tissue. Antibiotic toxicity and systemic side effects areserious problems associated with this course of therapy. Infectionsfollowing surgery, drug side effects, and related complications cansignificantly increase hospital stays and result in adverse outcomes.Because current methods for treating or preventing infection,particularly infections related to open fractures, are inadequate,improved compositions and methods for providing agents to prevent ortreat an infection at a site of trauma are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features chitosan compositionsthat provide for the delivery of biologically active agents for thetreatment or prevention of a pathogen infection. Advantageously, thedegradation and drug elution profiles of the chitosan compositions canbe tailored to the needs of particular patients at the point of care(e.g., in a surgical suite, clinic, physician's office, or otherclinical setting).

In one aspect, the invention features a method for producing abiodegradable chitosan composition having a desired biodegradationprofile, the method involving dissolving chitosan having a uniformdegree of deacetylation of at about 51% in one or more acids in asolvent, where the acid and the solvent are selected to produce achitosan that biodegrades over at least about one, two, three, four,five, six, seven, eight, nine, ten days or more in vivo; and forming thechitosan into a desired shape under conditions that reduce the watercontent by about 10%-100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%). In one embodiment, the method further involves contactingthe chitosan composition with an effective amount of at least one agentand/or incorporating an effective amount of at least one agent into thechitosan composition at a point of care.

In another aspect, the invention features a method for producing abiodegradable chitosan composition having a desired biodegradationprofile, the method involving dissolving chitosan having a uniformdegree of deacetylation of at about 51% in one or more acids in asolvent, where the acid and the solvent are selected to produce achitosan that biodegrades over at least about one, two, three, four,five, six, seven, eight, nine, ten days or more in vivo; forming thechitosan into a desired shape under conditions that reduce the watercontent by about 10%-100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%); and neutralizing the chitosan composition by contacting thecomposition with water, a neutral, or a basic solution, where the water,neutral, or basic solution is selected to modulate a physical-mechanicalproperty of the chitosan, thereby producing a biodegradable chitosancomposition. In one embodiment, the method further involves contactingthe chitosan composition with an effective amount of at least one agentand/or incorporating an effective amount of at least one agent into thechitosan composition at a point of care.

In yet another aspect, the invention features a method for producing abiodegradable chitosan composition containing an agent selected by aclinician at a point of care, the method involving dissolving chitosanhaving a desired biodegradation profile in one or more acids in asolvent where the acid and the solvent are selected to produce achitosan that biodegrades over at least about one, two, three, four,five, six, seven, eight, nine, ten days or more in vivo; forming thechitosan into a desired shape under conditions that reduce the watercontent by about 10%-100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%); neutralizing the chitosan composition by contacting thecomposition with water, a neutral, or a basic solution, where the water,neutral, or basic solution is selected to modulate a physical-mechanicalproperty of the chitosan; thereby producing a biodegradable chitosancomposition; selecting an agent; and incorporating an effective amountof at least one agent into the chitosan composition at a point of care.In one embodiment, the composition is contacted with the agent at apoint of care. In one embodiment, the agent (e.g., antimicrobial) isselected based on the source of trauma.

In another aspect, the invention features a chitosan compositionproduced by the method of any of the above aspects.

In another aspect, the invention features a wound management devicecontaining a chitosan composition produced by the method of any ofclaims 1-21.

In one aspect, the invention features an acid-treated chitosancomposition that degrades within 1-35 days in vivo containing orconsisting essentially of chitosan having a uniform degree ofdeacetylation of at least about 51%, where the water content is about0-90%, and an effective amount of an agent selected at the point ofcare.

In another aspect, the invention features a wound management devicecontaining an acid-treated chitosan composition that degrades within1-35 days in vivo containing or consisting essentially of chitosanhaving a uniform degree of deacetylation of at least about 51%, wherethe water content is about 0-90%, and an effective amount of an agent(e.g., antimicrobial agent, growth factor, anti-inflammatory, clotpromoting agent, and/or anti-thrombotic).

In another aspect, the invention features a method for treating orpreventing an infection in a subject at a site of trauma (e.g., afracture, open fracture, wound, complex wound, and surgical site), themethod involving contacting the site with a wound management devicecontaining or consisting essentially of a chitosan composition producedaccording to any of claims 19 and 21-31 and an effective amount of atleast one agent selected at the point of care (e.g., a surgical suite,clinic, physician's office, or other clinical setting).

In another aspect, the invention features a method for treating orpreventing an infection in a subject at a site of trauma (e.g., afracture, open fracture, wound, complex wound, and surgical site), themethod involving contacting the site with a wound management devicecontaining an acid-treated chitosan composition, said compositioncontaining or consisting essentially of chitosan having a uniform degreeof deacetylation of at least about 61% and an effective amount of atleast one agent selected at the point of care.

In another aspect, the invention features a method for the localdelivery of an agent to a site of trauma (e.g., a fracture, openfracture, wound, complex wound, and surgical site), the method involvingcontacting the site with a chitosan composition containing an agentselected at the point of care, thereby delivering the agent to the site.In one embodiment, the method further involves irrigating and debridingthe site of trauma.

In another aspect, the invention features a medical device forimplantation containing an acid-treated chitosan composition having auniform degree of deacetylation of at least about 51% and furthercontaining an effective amount of an agent. In one embodiment, thechitosan composition is a film that adheres to the device. In anotherembodiment, the device contains titanium or stainless steel. In yetanother embodiment, the medical device is a prosthetic device.

In another aspect, the invention features a kit containing a chitosancomposition of any of claim 22 or 24-36 for use in treating a traumasite or delivering an agent. In one embodiment, the chitosan compositionis present in a wound management device or medical device forimplantation. In another embodiment, the chitosan composition is in theform of a plug, mesh, strip, suture, dressing, sponge, film, hydrogel,or combinations thereof.

In various embodiments of any of the above aspects, the chitosancomposition is in the form of a film, hydrogel, mesh, plug, strip,sponge, suture, dressing, or combinations thereof. In other embodimentsof any of the above aspects, the agent is any one or more of anantimicrobial (e.g., anti-bacterial, anti-viral, and anti-fungalagents), growth factor, anti-inflammatory, hemostatic, andanti-thrombotic. In other embodiments of any of the above aspects, theanti-bacterial agent is any one or more of daptomycin, vancomycin, andamikacin. In various embodiments of the invention delineated herein, thechitosan composition contains about 1 μg-500 mg (1, 5, 10, 50, 100, 200,300, 400, 500, 600, 700, 800, 900 μg; 1, 5, 10, 25, 50, 75, 100, 125,150, 200, 250, 300, 350, 400, 450, 500), 100-300 (100, 125, 150, 175,200, 225, 250, 275, 300 mg), or 200-300 (200, 300) mg antibiotic pergram chitosan. In various embodiments of any invention delineatedherein, a chitosan composition releases at least about 1 μg to 1 mg ofantibiotic per hour (e.g., 1, 5, 10, 25, 50, 75, 100, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000μg/hour). In various embodiments of the above aspects, or any aspect ofthe invention delineated herein, the chitosan composition releases about1 μg-50 mg (e.g., 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800,900 μg; 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50) of the agent in 12-72hours. In other embodiments of any invention delineated herein, thechitosan composition releases about 1 μg-500 mg of the agent per cm³sponge. In still other embodiments of any invention delineated herein,the chitosan composition releases about 15-40 mg in about 24 hours orreleases about 20 mg of agent in about 24 hours. In various embodimentsof the above aspects, or any aspect of the invention delineated herein,at least about 10-1000 μg (e.g., 10, 25, 50, 75, 100, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000 μg) ofthe agent is eluted from the device in one hour, twenty-four hours, orseventy two hours.

In still other embodiments of the above aspects or any other aspect ofthe invention delineated herein, the chitosan composition isbiodegradable over at least about one, two, three, four, five, six days,over one week, two weeks, three weeks, or over one, two, three or moremonths. In other embodiments, the chitosan is treated with an acid thatis any one or a combination of acetic, citric, oxalic, proprionic,ascorbic, hydrochloric, formic, salicylic and lactic acids. In variousembodiments of an invention delineated herein, the acid or acid solventcontains 1-99% (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100%) lactic and/or 1-99% (e.g.,1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 99, 100%) acetic acid. In one embodiment of the aboveaspects, the chitosan is treated with lactic acid, acetic acid, or acombination of those two. In still other embodiments of the invention,the acid solvent is a 0.05%, 1%, 2%, 3%, 4%, or 5% acid solutioncontaining a blend of 75% lactic acid and 25% acetic acid.

In various embodiments of an invention delineated herein, the chitosandegree of deacetylation, weight percent, neutralization solution,solvent make-up, and/or crystallinity is varied to customize thebiodegradation profile, elution profile, and/or a physical-mechanicalproperty of the chitosan composition. In various embodiments, thephysical-mechanical property is selected from the group consisting oftensile strength, Young's modulus, swelling, degradation, and acombination thereof.

In still other embodiments of the invention, the effective amount of theagent is sufficient to reduce the survival or proliferation of abacterial cell (e.g., Pseudomonas aeruginosa (lux) or Staphylococcusaureus). In still other embodiments of any of the aspects delineatedherein, the method reduces bacteria present at the site by at leastabout 20-100% (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100%) at 72 hours after contact with the chitosancomposition relative to an untreated control site. In still otherembodiments, the degree of deacetylation is at least about 61, 71, or 80percent or the composition contains a combination of chitosans eachhaving such a percent deacetylation. In still other embodiments of anyof the above aspects, chitosan percent deacetylation or weight percentis varied to customize the composition's degradation and elution rates.In still other embodiments, the acid is varied to customize thecomposition's degradation and elution rates. In yet other embodiments ofany of the above aspects, the composition is custom loaded with an agentby a clinician at the point of treatment. In still other embodiments,the device provides for the long term release of an agent. In onepreferred embodiment of the above aspects, the chitosan compositioncontains 61DDA or 71DDA chitosan that is neutralized with 0.175 or 0.5MNaOH. In one preferred embodiment, the chitosan composition is 80%deacetylated chitosan treated with lactic and/or acetic acid. In stillother embodiments, the desired shape is obtained by freezing thechitosan in a mold and lyophilizing the chitosan to form a sponge, or bypouring the chitosan into a thin layer and heating the chitosan to forma dehydrated chitosan film. In still other embodiments of the invention,the chitosan composition is molded to form a plug, mesh, strip, suture,dressing, sponge, or film. In yet other embodiments, the chitosancomposition contains a chitosan sponge in a chitosan gel, where thechitosan has a uniform degree of deacetylation of at least about 51%. Inone embodiment, at least about 25%-95% of the composite is a gelcomponent. In another embodiment, at least about 5%-75% of the compositeis sponge.

In various embodiments of a method delineated herein, the method is exvivo. In various embodiments of a method delineated herein, the chitosandegree of deacetylation, weight percent, neutralization solution,solvent make-up, and/or crystallinity is varied to customize thebiodegradation profile, elution profile, and/or a physical-mechanicalproperty of the chitosan composition.

In various embodiments of a method delineated herein, the method furtherinvolves neutralizing the chitosan composition by contacting thecomposition with water, a neutral, or a basic solution. In still otherembodiments, the invention further involves reducing the water contentby at least about 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%). In yet other embodiments of a method delineated herein, thechitosan composition biodegrades over at least about three-five dayswhen implanted in a subject. In various embodiments of a method of anyof the above aspects or any other method of the invention delineatedherein, the method further involves irrigating and debriding the site oftrauma.

The invention provides compositions featuring chitosan and methods forusing such compositions for the local delivery of biologically activeagents to an open fracture, complex wound or other site of infection.Compositions and articles defined by the invention were isolated orotherwise manufactured in connection with the examples provided below.Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

DEFINITIONS

By “chitosan” is meant a chitin-derived polymer that is at least 20%deacetylated. Preferably, chitosan is at least about 50% deacetylated.Chitin is a linear polysaccharide consisting of (1-4)-linked2-acetamido-2-deoxy-b-D-glucopyranose. Chitosan is a linearpolysaccharide consisting of (1-4)-linked2-amino-2-deoxy-b-D-glucopyranose. An exemplary chitosan polymer isshown in FIG. 15.

By “composite” is meant a mixture of materials. In one embodiment, acomposite comprises sponge fragments dispersed within a hydrogel.

By “acid treated chitosan” is meant chitosan that is solubilized in anacidic solution.

By “degrades” is meant physically or chemically breaks down in whole orin part. Preferably, the degradation represents a physical reduction inthe mass by at least about 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95% or100%.

By “film” is meant a thin layer of material.

By “long term release” is meant elution of an agent over the course oftwenty-four-seventy two hours or longer. By “sponge” is meant athree-dimensional porous matrix.

By “wound management device” or “wound healing device” is meant anymaterial used to protect or promote healing at a site of trauma.

By “agent” is meant any small compound, antibody, nucleic acid molecule,or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “alteration” is meant a change (increase or decrease) in theexpression levels or activity of a gene or polypeptide as detected bystandard art known methods such as those described herein. As usedherein, an alteration includes a 10% change in expression levels,preferably a 25% change, more preferably a 40% change, and mostpreferably a 50% or greater change in expression levels.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a chitosan analogretains the biological activity of a corresponding reference chitosanpolymer (e.g., manufactured chitosan), while having certain biochemicalmodifications that enhance the analog's function relative to a referencechitosan polymer. Such biochemical modifications could increase theanalog's ability to be degraded, to uptake or elute a therapeutic agent,or to increase or decrease mechanical strength.

By “antimicrobial” is meant an agent that inhibits or stabilizes theproliferation or survival of a microbe. In one embodiment, abacteriostatic agent is an antimicrobial. In other embodiments, anyagent that kills a microbe (e.g., bacterium, fungus, virus) is anantimicrobial.

By “anti-inflammatory” is meant an agent that reduces the severity orsymptoms of an inflammatory reaction in a tissue. An inflammatoryreaction within tissue is generally characterized by leukocyteinfiltration, edema, redness, pain, and/or neovascularization.Inflammation can also be measured by analyzing levels of cytokines orany other inflammatory marker.

By “biodegradable” is meant susceptible to breakdown by biologicalactivity. For example, biodegradable chitosan compositions aresusceptible to breakdown by enzymes present in vivo (e.g., lysozyme,N-acetyl-o-glucosaminidase and lipases). Degradation of a chitosancomposition of the invention need not be complete. A chitosancomposition of the invention may be degraded, for example, by thecleavage of one or more chemical bonds (e.g., glycosidic bonds).

By “clinician” is meant any healthcare provider. Exemplary cliniciansinclude, but are not limited to, doctors, veterinarians, osteopaths,physician's assistants, emergency medical technicians, medics, nursepractitioners, and nurses.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “customize” is meant tailor to suit the needs of a particularsubject.

By “degradation rate” is meant the time required to substantiallydegrade the composition. A composition is substantially degraded whereat least about 75%, 85%, 90%, 95% or more has been degraded. Methods formeasuring degradation of chitosan are known in the art and includemeasuring the amount of a sponge, film, composite or other compositionof the invention that remains following implantation in a subject orfollowing in vitro exposure to an enzyme having chitosan-degradingactivity.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ. In oneexample, a disease is a bacterial or other infection present in a woundsite. In another embodiment, a disease is sepsis.

By “effective amount” is meant the amount of an agent required toameliorate the symptoms of a disease relative to an untreated patient.The effective amount of active agent(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “elution rate” is meant the time required for an agent to besubstantially released from a composition. Elution can be measured bydetermining how much of an agent remains within the composition or bymeasuring how much of an agent has been released into the composition'ssurroundings. Elution may be partial (10%, 25%, 50%, 75%, 80%, 85%, 90%,95% or more) or complete. In one preferred embodiment, the agentcontinues to be released at an effective level for at least about 3, 4,5, 6, 7, 8, 9, or 10 days.

The invention provides a number of targets that are useful for thedevelopment of highly specific drugs to treat or a disordercharacterized by the methods delineated herein. In addition, the methodsof the invention provide a facile means to identify therapies that aresafe for use in subjects. In addition, the methods of the inventionprovide a route for analyzing virtually any number of compounds foreffects on wound healing or pathogen infection described herein withhigh-volume throughput, high sensitivity, and low complexity.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA,shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof,that when administered to a mammalian cell results in a decrease (e.g.,by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a targetgene. Chitosan compositions are useful for the delivery ofpolynucleotides, such as inhibitory nucleic acid molecules, useful forthe treatment or prevention of pathogen infection and related disease.Typically, a nucleic acid inhibitor comprises at least a portion of atarget nucleic acid molecule, or an ortholog thereof, or comprises atleast a portion of the complementary strand of a target nucleic acidmolecule. For example, an inhibitory nucleic acid molecule comprises atleast a portion of any or all of the nucleic acids delineated herein.

By “infection” is meant the presence of one or more pathogens in atissue or organ of a host. An infection includes the proliferation of amicrobe (e.g., bacteria, viruses, fungi) within a tissue of a subject ata site of trauma.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “modulate” is meant alter (increase or decrease). Such alterationsare detected by standard art known methods such as those describedherein.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

By “physical interaction” is meant an association that does not requirecovalent bonding. In one embodiment, a physical interaction includesincorporation into a chitosan composition of the invention.

By “point of treatment” is meant the site where healthcare is delivered.A “point of treatment” includes, but is not limited to, a surgicalsuite, physician's office, clinic, or hospital.

By “polymer” is meant a natural or synthetic organic molecule formed bycombining smaller molecules in a regular pattern.

By “profile” is meant a set of characteristics that define a compositionor process. For example, a “biodegradation profile” refers to thebiodegradation characteristics of a composition. In another example, an“elution profile” refers to elution characteristics of a composition.

By “prosthetic device” is meant an implanted medical device thatsubstitutes for or supplements a missing or defective part of the body.

By “small molecule” is meant any chemical compound.

By “trauma” is meant any injury that damages a tissue or organ of asubject. The injury need not be severe. Therefore, a trauma includes anyinjury that breaks the skin.

By “modulation” is meant any alteration (e.g., increase or decrease) ina biological function or activity.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By “uniform degree of deacetylation” refers to a chitosan compositionmade from a single type of chitosan, (e.g., 61DDA, 71DDA, or 81DDA). Inone embodiment, a chitosan composition having a uniform degree ofdeacetylation excludes chitosan compositions having a combination oftypes chitosans, where the chitosans have different degrees ofdeacetylation.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

By “reference” is meant a standard or control condition.

By “siRNA” is meant a double stranded RNA. Optimally, an siRNA is 18,19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhangat its 3′ end. These dsRNAs can be introduced to an individual cell orto a whole animal; for example, they may be introduced systemically viathe bloodstream. Such siRNAs are used to downregulate mRNA levels orpromoter activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are graphs showing antibiotic (amikacin/vancomycin) elutionfrom sterilized chitosan.

FIGS. 2A-2C show chitosan sponges. FIG. 2A shows a dehydrated spongeprior to re-hydration. FIG. 2B shows in situ re-hydration of a chitosansponge (average re-hydration is 6.5 ml). FIG. 2C shows a dehydrated andre-hydrated chitosan sponge after 1 minute in solution.

FIGS. 3A-3D show results of Zone of Inhibition (ZOI) studies performedwith sterile chitosan sponges. The far-left image of each figure (FIGS.3A-D) is a sponge soaked in sterile saline and the 2nd and 3rd image ineach figure is a sponge soaked in 5 mg/ml antibiotic solution. FIGS. 3Aand 3B are the 24 hour and 48 hour ZOI images, respectively, for spongessoaked in amikacin against Pseudomonas aeruginosa. FIGS. 3C and 3D arethe 24 hour and 48 hour ZOI images, respectively, for sponges soaked invancomycin against Staphylococcus aureus. The measured radial ZOI islisted under each image in millimeters.

FIGS. 4A-4D are graphs showing bacterial cell quantity in a goat opentibial fracture model. FIG. 4A shows results with Psuedomonasaeruginosa. Values listed in 6-post and 48-pre are percentages of theoriginal 6-hour pre-irrigation and debridement levels listed in thefirst column labeled 6-pre. (values given are mean±standard error of themean; n=5). FIG. 4B is a graphical representation of bacterial cellquantity (Staphylococcus aureus) in a goat open tibial fracture model.Values listed in 6-post and 48-pre are percentages of the original6-hour pre-irrigation and debridement levels listed in the first columnlabeled 6-pre. (values given are mean±standard error of the mean; n=5for No Tx group, n=4 for sponge Tx group) FIG. 4C shows results withPseudomonas aeruginosa and FIG. 4D shows results with Staphylococcusaureus are graphs showing bacterial cell quantity (in a goat open tibialfracture model in control and amikacin or vancomycin treatment groups.Values listed are percentages of the original 6-hour pre-irrigation anddebridement levels listed in the first column labeled 6-pre. (valuesgiven are mean±standard error of the mean; n=5 for No Tx group, n=4 forsponge Tx group) or are cell counts. (values given are mean±standarderror of the mean; n=5). In each of FIGS. 4A-4D, 48 hr-pre means 48hours post inoculation and 42 hours post treatment.

FIGS. 5A-5D are images showing bacteria levels in a tibial fracturemodel in goats. FIG. 5A shows bacterial levels 6 hour post-injury,post-inoculation, pre-irrigation and debridement. (goat 840—SP Tx) forStaphylococcus aureus. FIG. 5B shows 6 hour post-injury,post-inoculation, post-irrigation and debridement. Pre-sponge Tx (goat840—SP Tx) for Staphylococcus aureus. A comparison of FIGS. 5A and 5Bshow 74% bacteria reduction (S.a.) 48 hour post-injury,post-inoculation, post-irrigation and debridement. (goat 94—No Tx)[Staphylococcus aureus]. FIG. 5C shows 48 hour post-injury,post-inoculation, post-irrigation and debridement. 42 hr sponge Tx (goat840—SP Tx) [Staphylococcus aureus]. FIG. 5D shows 48 hour post-injury,post-inoculation, post-irrigation and debridement. (goat 94—No Tx)[Staphylococcus aureus. A comparison of In FIG. 5C the bacteria countobserved after treatment with an antibiotic-loaded chitosan sponge(S.a.) is 0%. In contrast, FIG. 5D shows 357% bacteria count reboundwith no treatment (S.a.).

FIG. 6 provides a graphical representation of antibiotic levels found ingoat serum during animal testing. Blood was drawn at 6, 12, 24, 36, and42 hours post-operation. Serum antibiotic levels were measured by TDx.(amikacin and vancomycin). Values listed are μg/ml+−std. dev) (n=5 foramikacin samples and n=4 for vancomycin). left column, amikacin; rightcolumn, vancomycin.

FIG. 7 is a graph that shows antibiotic uptake results. Daptomycin isabbreviated as D, vancomycin as V, and neither as N; the numbers 61, 71,and 80 are used to indicate the % degree of deacetylation; the acidsolvents, lactic acid and acetic acid, are abbreviated LA and HAc,respectively. Daptomycin with lactic acid and vancomycin with aceticacid film variations had the highest antibiotic uptake values. Theresults are represented as the average±standard deviation (*,p≧0.7769; * vs. all others, **, †, ††, p<0.0001).

FIG. 8 is a graph that indicates the swelling ratio of the chitosan filmvariations after one minute rehydration by in situ loading. n=6measurements for all groups. D, LA and V, HAc film variations had ahigher swelling ratio. The results are represented as theaverage±standard deviation (*, p=0.8381; * vs. all others except **,p<0.0001).

FIG. 9 is a graph showing ultimate tensile strength (UTS) results. As ameasure of strength, UTS indicated the stress at which a dehydratedchitosan film specimen broke in MPa. The ultimate tensile strength ofdehydrated chitosan films reported in stress, (*, p=0.2131; * vs. allothers, p<0.0001). Film variations 71HAc and 80HAc were similar and hadsignificantly higher UTS values than other variations. n=6 measurementsfor all groups.

FIG. 10 is a graph quantitating elasticity results. The Young's Modulusindicated the ratio of stress to strain in MPa. Results are shown as theaverage±standard deviation (**, p=0.6597; ** vs. all others, p<0.0001).Film variations 71HAc and 80HAc were similar and had significantlyhigher Young's modulus than all other variations. n=6 measurements forall groups.

FIGS. 11A and 11B are graphs showing elution of daptomycin andvancomycin, respectively, from in situ loaded chitosan films representedas the average±standard deviation. Both antibiotic release profiles for71% DDA films showed a bell shaped release. n=3 measurements for allgroups.

FIG. 12 shows a universal testing machine that is used to measure thestrength required to pull implant components or fixtures apart.Cylindrical adhesive testing fixtures are shown positioned in the gripsof the universal tensile machine with hydrated chitosan film in positionbetween the adhesive fixtures.

FIG. 13 is a graph showing the results of adhesion testing of chitosanfilms. Adhesion testing measures adhesive strength, i.e. the maximumtensile load per area, in kPa. The adhesive strength of chitosan filmsrehydrated for 1 min to titanium and stainless steel alloy substrateswas used to measured. Adhesive strength is represented as theaverage±standard deviation (* vs. all others, p≦0.0310). These resultsindicated that 61% and 80% DDA, lactic acid film variations on SS, and71% DDA, lactic acid films on Ti variations have the highest adhesivestrength. 71LA films on titanium alloy fixtures had significantly higheradhesive strength than all other variations except for SS61LA andTi71LA. n=6 measurements for all groups. left column, titanium; rightcolumn, stainless steel.

FIGS. 14A and 14B show the degradation of chitosan films with andwithout antibiotics. FIG. 14A shows lysozyme-mediated degradation of afilm in situ loaded with daptomycin. FIG. 14B shows lysozyme-mediateddegradation of a film in situ loaded with vancomycin. Each of these isrepresented as the average±standard deviation (*p=0.0243; **, p=0.0300;†, p=0.0133; ††, p<0.0001). At 80 hours and on, 71% and 80% DDA filmsshowed a significant decrease in degradation rate. n=5 measurements forall groups.

FIGS. 15A and 15B show the antibiotic activity of elution samples. FIG.15A shows antibiotic activity of daptomycin elution samples. FIG. 15Bshows antibiotic activity of vancomycin elution samples. Activity isindicated by the percent inhibition of S. aureus growth and representedas the average±standard deviation. All variation samples over 72 hrs hadnearly complete inhibition. n=3 measurements for all groups.

FIG. 16 shows the molecular structure of a chitosan polymer. Rfunctional groups are either H (deacetylated) or COCH3 (acetylated)units.

FIG. 17 is a graph showing the release of antibiotics from chitosansponges. The chitosan sponges contained only 1 antibiotic per sponge(i.e., loaded with amikacin alone, vancomycin alone, and daptomycinalone). Amikacin and vancomycin release showed similar profilesthroughout the duration of the study whereas daptomycin release wassignificantly less than the other two tested antibiotics by the 72 hrtimepoint. Concentrations are given in micrograms per milliliter (20 mlPBS used as elution medium). A table is provided with the concentrationmeasurements. The 1^(st) group contained amikacin only, the 2^(nd) groupcontained vancomycin only, and the 3^(rd) group contained daptomycinonly. Solutions containing 5 mg/ml effective concentration of amikacin,vancomycin, or daptomycin was used to hydrate the chitosan sponges.(n=3).

FIG. 18 is a graph showing the release of amikacin from chitosansponges. Vancomycin nor daptomycin affected the release of amikacin.Concentrations given in micrograms per milliliter (20 ml PBS used aselution medium). A table is provided with the concentrationmeasurements. The 1^(st) group was amikacin only, the 2^(nd) groupcontained amikacin+vancomycin, and the 3^(rd) group containedamikacin+daptomycin. Solutions containing 5 mg/ml effectiveconcentration of amikacin, amikacin+vancomycin, or amikacin+daptomycinwere used to hydrate the chitosan sponges. (n=3).

FIG. 19 is a graph showing the release of vancomycin from chitosansponges. Amikacin did not affect the release of vancomycin.Concentrations given in micrograms per milliliter (20 ml PBS used aselution medium). A table is provided with the concentrationmeasurements. The 1^(st) group was vancomycin only and the 2^(nd) groupcontained vancomycin+amikacin. Solutions containing 5 mg/ml effectiveconcentration of vancomycin or vancomycin+amikacin were used to hydratethe chitosan sponges. (n=3).

FIG. 20 is a graph showing the release of daptomycin from chitosansponges. Amikacin did not affect the release of daptomycin.Concentrations given in micrograms per milliliter (20 ml PBS used aselution medium). A table is provided with the concentrationmeasurements. The 1^(st) group contained daptomycin only and the 2^(nd)group contained daptomycin+amikacin. Solutions containing 5 mg/mleffective concentration of daptomycin or daptomycin+amikacin were usedto hydrate the chitosan sponges. (n=3).

FIGS. 21A and 21B are images depicting a chitosan sponge implantedadjacent to the catheter surface subcutaneously in the hind limb of amouse. The chitosan sponge was implanted immediately prior toimplantation of the catheter segment. Injection of 100 μl of 10⁵ CFU ofbacterial inoculum (USA300, CA-MRSA) was done into the lumen of thecatheter.

FIG. 22 is a graph showing catheters retrieved from the subjects withdaptomycin-loaded and vancomycin-loaded chitosan sponges had no bacteriapresent in all retrieved implants. Colony forming units per retrievedcatheter within each treatment type are graphed. \ The groups containinglinezolid-loaded and PBS-loaded chitosan sponges as well as the catheteronly controls all had colonized bacteria present on the implant. Thedaptomycin- and vancomycin-loaded chitosan sponge groups werestatistically significant from the other 3 groups in terms ofcfu/catheter (p<0.0001). The other 3 groups were not statisticallysignificant from each other. (n=12)

DETAILED DESCRIPTION OF THE INVENTION

As described below, the present invention features chitosan compositions(e.g., solids, sponges, films, hydrogels, composites) that provide forthe local delivery of biologically active agents and methods of usingsuch compositions to treat or prevent an infection or promote healing.

The invention is based, at least in part, on the discovery thatacid-treated chitosan can form wound management devices whosedegradation and drug elution properties can be customized to suit theneeds of specific subjects.

Chitosan

Chitosan is a naturally occurring linear polysaccharide composed ofrandomly distributed β-(1-4)-2-amino-2-D-glucosamine (deacetylated) andβ-(1-4)-2-acetamido-2-D-glucoseamine (acetylated) units (FIG. 15).Chitosan is derived from chitin, a naturally occurring polymer. Chitinis a white, hard, inelastic, nitrogenous polysaccharide isolated fromfungi, mollusks, or from the exoskeletons of arthropods (e.g.,crustaceans, insects). The major procedure for obtaining chitosan is thealkaline deacetylation of chitin with strong alkaline solution.Generally, the raw material is crushed, washed with water or detergent,and ground into small pieces. After grinding, the raw material istreated with alkali and acid to isolate the polymer from the raw crushedmaterial. The polymer is then deacetylated by treatment with alkali.Chitin and chitosan differ in their degrees of deacetylation (DDA).Chitin has a degree of deacetylation of 0% while pure chitosan has adegree of deacetylation of 100%. Typically, when the degree ofdeacetylation is greater than about 50% the polymer is referred to aschitosan.

Chitosan is a cationic weak base that is substantially insoluble inwater and organic solvents. Typically, chitosan is fairly soluble indilute acid solutions, such as acetic, citric, oxalic, proprionic,ascorbic, hydrochloric, formic, and lactic acids, as well as otherorganic and inorganic acids. Chitosan's charge gives it bioadhesiveproperties that allow it to bind to negatively charged surfaces, such asbiological tissues present at a site of trauma or negatively chargedimplanted devices. Chitosan's degree of deacetylation affects itresorption. Chitosan compositions having a 50% degree of deacetylationare highly degradable in vivo. As the degree of deacetylation increases,chitosan becomes increasingly resistant to degradation. Chitosancompositions having a degree of deacetylation that is higher than 95%degrade slowly over weeks or months. In the body chitosan is degraded bylysozyme, N-acetyl-o-glucosaminidase and lipases. Lysozyme degradeschitosan by cleaving the glycosidic bonds between the repeating chitosanunits. The byproducts of chitosan degradation are saccharides andglucosamines that are gradually absorbed by the human body. Therefore,when chitosan is used for the local delivery of therapeutic orprophylactic agents, no secondary removal operation is required.

As reported herein, chitosan compositions (e.g., solids, sponges, films,hydrogels, composites) can be loaded with a biologically active agent atthe site of care (e.g., in a surgical suite, clinic, or physician'soffice, trauma site, battlefield). This property allows the clinician totailor the antibiotics or other agents used to load the chitosan woundmanagement device to suit the needs of a particular patient. In oneembodiment, the degree of deacetylation is adjusted to provide chitosancompositions that degrade in as little as about twenty-four, thirty-six,forty-eight, or seventy two hours or that are maintained for a longerperiod of time (e.g., 4, 5, 6, 7, 8, 9, 10 days). In other embodiments,chitosan compositions of the invention are maintained in the body for atleast about two-six weeks or more (e.g., 2, 3, 4, 5, 6 weeks, two, threeor four months). In still other embodiments, chitosan compositions ofthe invention enhance blood clotting in a wound or other site of trauma(hemostasis). In other embodiments, the chitosan compositions are loadedwith therapeutic or prophylactic agents that are clinician selected andthat are delivered over at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10days or for longer periods.

Antimicrobial Agents

Staphylococcus aureus, staphylococcus epidermidis, and Pseudomonasaeruginosa are pathogens that are commonly present at musculoskeletalwound sites. S. aureus is one cause of osteomyelitis and nongonococcalbacterial arthritis, and is often associated with prosthetic jointinfection. The invention provides chitosan compositions useful intreating or preventing infection in a wound, complex wound, openfraction, or other site of trauma. Any antimicrobial agent known in theart can be used in the chitosan compositions of the invention atconcentrations generally used for such agents.

Antimicrobial agents useful in chitosan compositions of the inventioninclude but are not limited to antibacterials, antifungals, andantivirals. An antimicrobial agent as used herein is an agent whichreduces or stabilizes the survival, growth, or proliferation of apathogen. Antimicrobial agents include but are not limited to Aztreonam;Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane; PirazmonamSodium; Propionic Acid; Pyrithione Sodium; Sanguinarium Chloride;Tigemonam Dicholine; Acedapsone; Acetosulfone Sodium; Alamecin;Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin;Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; AmpicillinSodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium;Bacampicillin Hydrochloride; Bacitracin; Bacitracin MethyleneDisalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; BiphenamineHydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate;Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; CarbenicillinIndanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium;Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; CefepimeHydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride;Cefinetazole; Cefinetazole Sodium; Cefonicid Monosodium; CefonicidSodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan;Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium;Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium;Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine;Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium;Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; CephalexinHydrochloride, Cephaloglycin; Cephaloridine; Cephalothin Sodium;Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol PantothenateComplex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycinlydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacil; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz: Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium: TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin;Zorbamycin; Difloxacin Hydrochloride; Lauryl Isoquinolinium Bromide;Moxalactam Disodium; Ornidazole; Pentisomicin; and SarafloxacinHydrochloride. In particular embodiments, a chitosan compositioncomprises daptomycin.

In one preferred embodiment, a chitosan composition of the inventioncomprises an agent that treats a multidrug resistant bacteria. In oneapproach, linezolid may be used to treat multi-drug resistant Grampositive bacteria. Linezolid is commercially available under the tradename Zyvox (Pfizer).

In other embodiments, a chitosan composition comprises one or more ofthe following: Benzalkonium Chloride, Cetylpyridinium Chloride, andChlorhexidine Digluconate. In still other embodiments, a chitosancomposition comprises one or more of antimicrobials: PolyhexamethyleneBiguanide, Octenidine Dihydrochloride, Mild Silver Protein, PovidoneIodine (solution or ointment), Silver Nitrate, Silver Sulfadiazine,Triclosan, Cetalkonium Chloride, Myristalkonium Chloride, Tigecycline,Lactoferrin, Quinupristin/dalfopristin, Linezolid, Dalbavancin,Doripenem, Imipenem, Meropenem, and Iclaprim.

In still other embodiments, the chitosan composition comprises anessential oil having antimicrobial properties. Exemplary essential oilsinclude Oregano oil, tea tree oil, mint oil, sandalwood oil, clove oil,nigella sativa oil, onion oil, leleshwa oil, lavender oil, lemon oil,lemon myrtle oil, neem oil, garlic, eucalyptus oil, peppermint oil,cinnamon oil, and thyme oil.

In still other embodiments, the antimicrobial is a fatty acid (e.g.,Cis-2-Decenoic Acid).

Antivirals are agents capable of inhibiting the replication of viruses.Examples of anti-viral agents include but are not limited to1,-D-ribofuranosyl-1,2,4-triazole-3 carboxamide, 9-2-hydroxy-ethoxymethylguanine, adamantanamine, 5-iodo-2′-deoxyuridine,trifluorothymidine, interferon, adenine arabinoside, proteaseinhibitors, thymidine kinase inhibitors, sugar or glycoprotein synthesisinhibitors, structural protein synthesis inhibitors, attachment andadsorption inhibitors, and nucleoside analogues such as acyclovir,penciclovir, valacyclovir, and ganciclovir.

Antifungal agents useful in chitosan compositions of the inventioninclude fungicidal and fungistatic agents such as, for example, benzoicacid, undecylenic alkanolamide, ciclopirox olamine, polyenes,imidazoles, allylamine, thicarbamates, amphotericin B, butylparaben,clindamycin, econaxole, fluconazole, flucytosine, griseofulvin,nystatin, and ketoconazole. In one preferred embodiment, the antifungalis amphotericin.

In one embodiment, the invention provides chitosan compositionscomprising a combination of one or more antimicrobials and antivirals orantifungals.

Growth Factors

Growth factors are typically polypeptides or fragments thereof thatsupport the survival, growth, or differentiation of a cell. Such agentsmay be used to promote wound healing. A chitosan composition describedherein can be used to deliver virtually any growth factor known in theart. Such growth factors include but are not limited to angiopoietin,acidic fibroblast growth factors (aFGF) (GenBank Accession No.NP_(—)149127) and basic FGF (GenBank Accession No. AAA52448), bonemorphogenic protein (BMP) (GenBank Accession No. BAD92827), vascularendothelial growth factor (VEGF) (GenBank Accession No. AAA35789 orNP_(—)001020539), epidermal growth factor (EGF) (GenBank Accession No.NP_(—)001954), transforming growth factor cc (TGF-α) (GenBank AccessionNo. NP_(—)003227) and transforming growth factor β (TFG-β) (GenBankAccession No. 1109243A), platelet-derived endothelial cell growth factor(PD-ECGF) (GenBank Accession No. NP_(—)001944), platelet-derived growthfactor (PDGF) (GenBank Accession No. 1109245A), tumor necrosis factor cc(TNF-α) (GenBank Accession No. CAA26669), hepatocyte growth factor (HGF)(GenBank Accession No. BAA14348), insulin like growth factor (IGF)(GenBank Accession No. P08833), erythropoietin (GenBank Accession No.P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF)(GenBank Accession No. AAB59527), granulocyte/macrophage CSF (GM-CSF)(GenBank Accession No. NP_(—)000749) and nitric oxide synthase (NOS)(GenBank Accession No. AAA36365). In one preferred embodiment, thegrowth factor is BMP.

Analgesics

Chitosan compositions of the invention can be used for the delivery ofone or more agents that ameliorate pain, such agents include but are notlimited to opioid analgesics (e.g. morphine, hydromorphone, oxymorphone,levorphanol, levallorphan, methadone, meperidine, fentanyl, codeine,dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene,nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphineor pentazocine; a nonsteroidal antiinflammatory drug (NSAID) (e.g.,aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen,flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen,ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen,oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac,or a pharmaceutically acceptable salt thereof; a barbiturate sedative,e.g. amobarbital, aprobarbital, butabarbital, butabital, mephobarbital,metharbital, methohexital, pentobarbital, phenobartital, secobarbital,talbutal, theamylal or thiopental or a pharmaceutically acceptable saltthereof; a COX-2 inhibitor (e.g. celecoxib, rofecoxib or valdecoxib.

Anti-Thrombotic

Chitosan compositions of the invention are also useful for inhibiting,reducing or ameliorating clot formation. In one embodiment, a chitosancomposition contains one or more anti-thrombotids (e.g., thrombin,fibrinogen, cumidin, heparin).

Anti-Inflammatories

In other embodiments, a chitosan composition is used to deliver ananti-inflammatory agent. Such anti-inflammatory agents include, but arenot limited to, Alclofenac; Alclometasone Dipropionate; AlgestoneAcetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium;Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;Desonide; Desoximetasone; Dexamethasone Dipropionate; DiclofenacPotassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; and Zomepirac Sodium.

Delivery of Agents Via Chitosan Compositions

The invention provides a simple means for delivering biologically activeagents (e.g., small compounds, nucleic acid molecules, polypeptides)using a chitosan composition. The chitosan composition is delivered to asubject and the biologically active agent is eluted from the compositionin situ. The chitosan composition is capable of delivering a therapeuticfor the treatment of a disease or disorder that requires controlledand/or localized drug delivery over some period of time (e.g., 1, 3, 5,7 days; 2, 3, 4 weeks; 1, 2, 3, 6, 12 months). Desirably, the chitosancomposition comprises an effective amount of one or more antibiotics(e.g., amikacin, daptomycin, vancomycin), growth factors that promotewound healing, small molecules, hemostatic agents (e.g., thrombin and/orfibrinogen), anti-thrombotics (e.g., heparin), or cartilage or bonerepair agents. The chitosan composition are administered in the form ofsolids, sponges, films, hydrogels, or composites (e.g., sponge fragmentsin a hydrogel matrix).

Preferably, the chitosan composition comprises at least about 1 μg, 25μg, 50 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1 mg, 5 mg, 10 mg, 25 mg, 50mg, 75 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg of an agent(e.g., an antibiotic). In another embodiment, the composition releasesat least about 1 μg, 25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1 mg,5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400mg, or 500 mg of an agent (e.g., an antibiotic) over the course of atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 21, 28, or 35 days. Instill another embodiment, the composition comprises at least about 1 μg,25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1 mg, 5 mg, 10 mg, 25 mg,50 mg, 75 mg, 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg of anagent (e.g., an antibiotic) per cm³.

Chitosan Coatings

A chitosan composition may be included in a coating material, such as afilm, that is used to coat or wrap a medical device (e.g., drug deliveryor other medical device). Such coatings are used, for example, fortreating or preventing a pathogen infection or for drug delivery. Inorthopaedics, many post-surgical infections are associated with implantmaterials. Patients receiving an orthopedic implants have an infectionrisk of about 5% for total joint replacements. Bacteria are passivelyadsorbed on biomaterial surfaces after implantation. The fundamentalpathogenic mechanism in biomaterial-centered sepsis is microbialcolonization of the biomaterials followed by adjacent damaged tissues.Patients that suffer from such infections often require the removal andreplacement of the implant to eradicate the infection.

To treat or prevent an implant-associated infection a chitosancomposition of the invention is applied to the medical device (e.g.,implant). The chitosan composition provides for release of a therapeuticor prophylactic agent from the device. Such agents advantageously reducethe risk of infection associated with conventional implants. Suchcoatings can be applied to any medical device known in the art,including, but not limited to orthopedic devices (e.g., for jointimplants, fracture repairs, spinal implants, screws, rods, plates);surgical devices (e.g., sutures, staples, anastomosis devices, vertebraldisks, bone pins, suture anchors, hemostatic barriers, clamps, screws,plates, clips, vascular implants, tissue adhesives and sealants, tissuescaffolds); wound management devices; drug-delivering vascular stents(e.g., a balloon-expanded stents); other vascular devices (e.g., grafts,catheters, valves, artificial hearts, heart assist devices); implantabledefibrillators; blood oxygenator devices (e.g., tubing, membranes);membranes; biosensors; shunts for hydrocephalus; endoscopic devices;infection control devices; dental devices (e.g., dental implants,fracture repair devices), urological devices (e.g., penile, sphincter,urethral, bladder and renal devices, and catheters); colostomy bagattachment devices; ophthalmic devices (e.g. intraocular coils/screws);glaucoma drain shunts; synthetic prostheses (e.g., breast); intraocularlenses; respiratory, peripheral cardiovascular, spinal, neurological,dental, ear/nose/throat (e.g., ear drainage tubes); renal devices; anddialysis (e.g., tubing, membranes, grafts), urinary catheters,intravenous catheters, small diameter grafts, vascular grafts,artificial lung catheters, atrial septal defect closures,electro-stimulation leads for cardiac rhythm management (e.g., pacerleads), glucose sensors (long-term and short-term), degradable coronarystents (e.g., degradable, non-degradable, peripheral), blood pressureand stent graft catheters, birth control devices, prostate cancerimplants, bone repair/augmentation devices, breast implants, cartilagerepair devices, dental implants, implanted drug infusion tubes,intravitreal drug delivery devices, nerve regeneration conduits,oncological implants, electrostimulation leads, pain managementimplants, spinal/orthopedic repair devices, wound dressings, embolicprotection filters, abdominal aortic aneurysm grafts, heart valves(e.g., mechanical, polymeric, tissue, percutaneous, carbon, sewingcuff), valve annuloplasty devices, mitral valve repair devices, vascularintervention devices, left ventricle assist devices, neuro aneurysmtreatment coils, neurological catheters, left atrial appendage filters,hemodialysis devices, catheter cuff, anastomotic closures, vascularaccess catheters, cardiac sensors, uterine bleeding patches, urologicalcatheters/stents/implants, in vitro diagnostics, aneurysm exclusiondevices, and neuropatches.

Examples of other suitable devices include, but are not limited to, venacava filters, urinary dialators, endoscopic surgical tissue extractors,atherectomy catheters, clot extraction catheters, coronary guidewires,drug infusion catheters, esophageal stents, circulatory support systems,angiographic catheters, coronary and peripheral guidewires, hemodialysiscatheters, neurovascular balloon catheters, tympanostomy vent tubes,cerebro-spinal fluid shunts, defibrillator leads, percutaneous closuredevices, drainage tubes, thoracic cavity suction drainage catheters,electrophysiology catheters, stroke therapy catheters, abscess drainagecatheters, biliary drainage products, dialysis catheters, central venousaccess catheters, and parental feeding catheters.

It is noted that in other embodiments of the present invention, thechitosan composition of the present invention may self adhere to themedical device or may be adhered to the device by means other thancoating materials, such as adhesives, sutures, or compression. Anysuitable method know in the art may be utilized to adhere the chitosancomposition to a surface. For example, the chitosan composition may beadhered to the surface by pressing the chitosan composition onto thedevice, wrapping the device with a chitosan film, or spraying a chitosancomposition onto the device.

The chitosan compositions with biocompatible surfaces may be utilizedfor various medical applications including, but not limited to, drugdelivery devices for the controlled release of pharmacologically activeagents, including wound healing devices, such as hemostatic sponges,dressings, suture material and meshes, medical device coatings/films andother biocompatible implants.

Chitosan Fibers

Randomly oriented fibrous mats can be made from chitosan byelectrospinning (Schiffman and Schauer, Biomacromolecules, 2007. 8(9):p. 2665-7). These fibers are typically a mean diameter of 100 nm, butthe diameter of the fibers can vary widely depending upon a number offactors. Some fibers can be as large as 1 μm in diameter, and as smallas 28 nm. Typically, fibrous mats have a mean diameter between 100-200nm.

In one approach, about 7-9 wt % chitosan is dissolved in 99-100%trifluoroacetic acid. The solvent solution contains about 0-30%methylene chloride to aid in spinnability. The solution is gently mixedfor 24 hours. At this point additives such as drugs, proteins, calciumphosphate salts, or other biologically active constituents can be added.The solution is loaded into a plastic 10 mL syringe. In one embodiment,a blunt 21 gauge (G) metal needle is used. In other embodiments, needlesizes range from about 16-23G. The syringe is loaded into a syringe pumpand the flowrate is set for 20-30 μL/min (usually 20 μL/min). The needleis connected to the positive electrode of the power source, while thetarget is connected to the ground. The target can consist of a copperplate wrapped in aluminum foil, an aluminum SEM stub, or any otherconductive surface. The voltage is set between 15-26 kV. The distancebetween the tip and the target is between about 12-25 cm. In onepreferred embodiment, 15-16 cm is used. Typically, the apparatus is usedinside a ventilated box within fume hood. The box protects the fibersfrom air currents as they are deposited on the target. Once deposited,the fiber mat can be removed from the surface and treated as follows.Typically, the fiber mat is maintained under vacuum for about 24 hours,and then the fiber mat is neutralized in 5M Na₂CO₃. After drying, matsare cut to any desired size and sterilized. Another method toelectrospin chitosan involves using 1,1,1,3,3,3-Hexafluoroisopropanol(HFIP) as a solvent (Shin et al., J Periodontol, 2005. 76(10): p.1778-84). One advantage is that there is no need to neutralize the mat.The residual solvent is pulled off in vacuum. HFIP solvent can also beused with methylene chloride to aid in spinning. Other methods forgenerating chitosan fibers are described, for example, by Sangsanoh andSupaphol, Biomacromolecules, 2006. 7(10): p. 2710-4 or Schiffman andSchauer, Biomacromolecules, 2007. 8(2): p. 594-601.

Wound Healing Devices

The present invention provides wound healing devices that employ achitosan composition. The wound healing devices may be configured byforming the chitosan composition into a shape and size sufficient toaccommodate the wound being treated. If desired, the wound healingdevice comprises chitosan fibers. Wound healing devices are desirablyproduced in whatever shape and size is necessary to provide optimumtreatment to the wound. These devices can be produced in forms thatinclude, but are not limited to, plugs, meshes, strips, sutures,dressings, or any other form able to accommodate and assist in therepair of a wound. The damaged portions of the patient that may betreated with devices made of the chitosan composition of the presentinvention include, but are not limited to, bone, cartilage, skin, muscleand other tissues (nerve, brain, spinal cord, heart, lung). Othersimilar devices are administered to assist in the treatment repair andremodeling of a damaged tissue, bone, or cartilage. For someapplications, it is desirable for the device to be incorporated into anexisting tissue to facilitate wound repair. For other applications, itis desirable for the device to degrade over the course of days, weeks,or months. Such degradation may be advantageously tailored to suit theneeds of a particular subject using the methods described herein. Theelution and/or degradation profile of a chitosan composition (e.g.,film, sponge) can be altered as described herein by modulating thefollowing variables: degree of deacetylation, neutralization solution,solvent make-up, and chitosan weight %, and/or crystallinity.

Crystallinity indicates the degree of structural order in a compound.Polymers such as chitosan are either amorphous or semicrystalline.Chitosan's crystallinity, like other polymers, depends on its type,number, and regularity of polymer-chain, side group chemistry, thedegree of matrix packing or density, and crosslinking. The crystallinityof chitosan or its products can be controlled or altered duringmanufacture through its molecular weight, degree of deacetylation, andcrosslinking to affect thermal properties, such as melting point, andphysical-mechanical properties, such as tensile strength, Young'smodulus, swelling and degradation.

Crosslinking is the process which links polymer chains together. Inchitosan, crosslinking induces a three-dimensional matrix ofinterconnected, linear, polymeric chains. The degree or extent ofcrosslinking depends on the crosslinking agent. Exemplary crosslinkingagents include sodium tripolyphosphate, ethylene glycol diglycidylether, ethylene oxide, glutaraldehyde, epichlorohydrin, diisocyanate,and genipin. Crosslinking can also be accomplished using microwave orultraviolet exposure.

Chitosan's properties can also be altered by modulating the degree ofdeacetylation. In one embodiment, the degree of deacetylation isadjusted between about 50-100%, wherein the bottom of the range is anyinteger between 50 and 99, and the top of the range is any integerbetween 51% and 100%. In particular embodiments, the degree ofdeacetylation is 51%, 55%, 60%, 61%, 65%, 70%, 71%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 85%, 90%, and 95%. In general, the higher themolecular weight, the slower the degradation of the chitosancomposition.

If desired, chitosan is neutralized after acid treatment. Any base knownin the art (e.g., NaOH, KOH, NH₄OH, Ca(OH)₂, Mg(OH)₂, or combinationsthereof) may be used to neutralize an acid-treated chitosan composition.Preferably, a neutralization solution has a pH greater than 7.4 (e.g.,7.8, 8.0, 8.5, 9.0, 10, 11, and 12, 13, 14, 15, 16). The neutralizationstep is optional, and not strictly required. If desired, the chitosan istreated with water, PBS, or sterile saline following acid treatment. Itmay comprise 0.01-10.0M of a base (e.g., 0.01, 0.025, 0.5, 0.75, 0.1,0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 M) (e.g.,NaOH). Chitosan compositions neutralized in bases having lower molaritydegrade more quickly. Chitosan compositions neutralized in bases ofincreased molarity degrade more slowly than those neutralized at lessermolarities. Thus, the degradation properties of chitosan can bemodulated by altering the molarity of the neutralizing base.

In other embodiments, the concentration of the acidic solvent used todissolve the chitosan is adjusted or the time period used to dissolvethe chitosan is altered. For example, a 0.1%, 0.5%, 1%, 2%, 3% or 5%acid solution is used. In particular embodiments, chitosan is dissolvedin acetic, citric, oxalic, proprionic, ascorbic, hydrochloric, formic,salicylic and/or lactic acids, or a combination of those. In general,acidic solvents comprising increased levels of lactic acid form chitosancompositions that degrade more quickly and also have reduced strengthand durability. In various embodiments, a combination of acetic andlactic acids are used. Acetic provides more strength and slowerdegradation. In contrast, lactic acid provides more flexibility. In oneapproach, the ratio of lactic to acetic acid is varied from 5:1, 4:1,3:1, 2:1, 1:1, 1:2, 1:3, 1:4, to 1:5. In one embodiment, the blendedacid solvent comprises 90%/10%, 80%/20% 75%/25%, 70%/30%, 60%/40%,50%/50%. In still other embodiments, the chitosan weight % is alteredfrom 0.25-10.0% (e.g., 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1, 1.25, 1.5,1.75, 2.0, 2.5, 3.0, 3.5, 4, 5, 6, 7, 8, 9, 10%). In one embodiment, a 1wt % chitosan solution is preferred, where a 1 wt % chitosan solutioncontains 1 gram of chitosan per 100 ml solution. Typically, the higherthe wt %, the slower the degradation.

If desired the chitosan composition is loaded with agents and thechitosan composition is delivered to a wound to form a delivery systemfor the agent. Preferably, the chitosan composition contains aneffective amount of a chemical or pharmaceutically active component. Inone embodiment, the chitosan composition self-adheres to a site at whichdelivery is desired. In another embodiment, an adhesive or otheradhering means may be applied to the outer edges of the chitosancomposition to hold the composition in position during the delivery ofthe chemical or pharmaceutically active component. Such adherent meansmay be used alone or in combination with the self-adhering properties ofchitosan. Chitosan compositions provide for the local administration ofa desired amount of a therapeutic agent.

Other embodiments of the present invention include wound-healing devicesconfigured and produced as biological fasteners, such as threads,sutures and woven sheets. Threads and sutures comprising variousembodiments of the chitosan composition provide a biocompatiblefastening and suturing function for temporarily treating and sealing anopen wound. Additionally, the biological fasteners may includepharmacologically active agents that may assist in the healing andremodeling of the tissue within and around the wound. Advantageously,such fastening and suturing devices may be treated to degrade in vivo ata desired rate. In other embodiments, the chitosan composition isadministered directly to an injured area. A chitosan composition of theinvention is administered by sprinkling, packing, implanting, insertingor applying or by any other administration means to a site of trauma(e.g., open wound, open fracture, complex wound).

Hemostatic Chitosan Compositions

The invention further provides chitosan compositions in the form of ahemostatic matrix (e.g., hemostatic sponges). Such compositions areuseful alone or may be used for the delivery of a therapeutic orprophylactic agent delineated herein. Such matrices generally compriseporous compositions formed from chitosan. In general, sponges can beformed by providing a liquid solution of chitosan capable of forming aporous three-dimensionally stable structure. In one embodiment, achitosan solution is prepared by dissolving deacetylated chitosan in anacidic solvent. A sponge is formed by casting the solution in a mold toachieve a desired shape. The chitosan solution is then frozen andlyophilized, thereby forming a chitosan sponge. Lyophilization isconducted to reduce the liquid (e.g. water) content of the matrix toless than about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, or100% by weight. If desired, a second lyophilization step is carried out.This step is strictly optional. Following one or more lyophilizations,the chitosan composition may still include some amount of water.Typically, lypholization removes at least about 70%, 75%, 80%, 90%, 95,or 100% or the original water content of the chitosan composition.Chitosan compositions that retain some moisture may be packaged insterile foil to maintain such moisture.

In one approach, the sponge is neutralized, for example, by treatmentwith a basic solution, re-lyophilized. The sponge matrix is stabilizedstructurally and remains in a highly dense and compacted state untilcontacted with a liquid susceptible to absorption by the matrix, forexample, body fluids. For medical use, the compacted or compressedsponge is sterilized using any suitable means (e.g., radiation). Thedevice is packaged in sterile packaging for medical use. Sponge elementsor other devices of the invention may also contain one or more activetherapeutic agents. For example, they include agents that promoteclotting (e.g., thrombin and/or fibrinogen). Alternatively or inaddition, sponge elements or other devices of the invention includeantibiotics and/or growth factors that promote tissue growth andhealing.

A chitosan composition is incubated with a therapeutic agent such thatthe agent is incorporated into the chitosan. This incubation istypically carried out before or during a procedure to treat a subjectusing methods described herein. Sponge materials of the invention willadvantageously be expandable when wetted. Preferably, the sponge has thecapacity to expand at least about 10%-100% (10, 20, 30, 40, 50). Inother embodiments, a sponge expands by about 200% by volume when wettedto saturation with deionized water, buffer, or an agent of theinvention. Preferred sponge materials achieve rapid volume expansions(e.g., when immersed in aqueous solution). Hemostatic sponges areproduced in any size required for application to a wound. In oneembodiment, the expanded sponge exerts compression on surroundingtissues when implanted or delivers an active agent to the implantationsite and surrounding tissue.

Delivery of Chitosan Compositions

Chitosan compositions can be delivered by any method known to theskilled artisan. In one approach, a chitosan composition is locallydelivered to a site of trauma in the form of a film or sponge. The film,sponge, or other wound management device can be configured to fit awound of virtually any size. In another approach, the chitosancomposition is surgically implanted at a site where promotion of healingand/or treatment or prevention of infection is required. If desired, thechitosan composition is loaded with one or more antibiotics or otherbiologically active agents by a clinician within the surgical suitewhere treatment is to be provided. This advantageously allows thechitosan composition to be loaded with a specific agent or combinationof agents tailored to the needs of a particular patient at the point atwhich care is to be provided.

Screening Assays

As described herein, the present invention provides for the delivery oftherapeutic or prophylactic agents to wounds in vivo. The invention isbased in part on the discovery that therapeutic agents can be deliveredusing a chitosan composition where the agents and degradation of thecomposition is tailored to suit the needs of a particular patient. Toidentify chitosan compositions having the desired degradation andelution profiles, screening may be carried out using no more thanroutine methods known in the art and described herein. For example,chitosan compositions are loaded with one or more therapeutic agents andsuch compositions are subsequently compared to untreated controlcompositions to identify chitosan compositions that promote healing. Inanother embodiment, the degradation of a chitosan composition of theinvention is assayed in vivo to identify the degree of deacetylationthat corresponds to a the desired degradation profile. Any number ofmethods are available for carrying out screening assays to identify suchcompositions.

In one working example, candidate compounds are added at varyingconcentrations to a chitosan composition. The degree of infection orwound healing is then measured using standard methods as describedherein. The degree of infection (e.g., number of bacteria) or woundhealing in the presence of the compound is compared to the levelmeasured in a control lacking the compound. A compound that enhanceshealing is considered useful in the invention; such a compound may beused, for example, as a therapeutic to prevent, delay, ameliorate,stabilize, or treat a disease described herein (e.g., tissue damage). Inother embodiments, the compound prevents, delays, ameliorates,stabilizes, or treats a disease or disorder described herein. Suchtherapeutic compounds are useful in vivo.

In another approach, chitosan compositions having varying degrees ofdeacetylation are incubated in vivo, added to a wound, or are contactedwith a composition comprising an enzyme having chitosan-degradingactivity. The length of time required for chitosan degradation is thenmeasured using standard methods as described herein. A chitosancomposition having the desired degradation profile (e.g., degrading in 3days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months) isconsidered useful in the invention; such a composition may be used, forexample, as a therapeutic to prevent, delay, ameliorate, stabilize, ortreat a disease described herein (e.g., tissue damage). In otherembodiments, the composition prevents, delays, ameliorates, stabilizes,or treats a disease or disorder described herein. Such therapeuticcompositions are useful in vivo.

The present invention provides methods of treating pathogen infections(e.g., bacterial, viral, fungal), complex wounds, open fractures,trauma, and associated diseases and/or disorders or symptoms thereofwhich comprise administering a therapeutically effective amount of acomposition comprising chitosan and a therapeutic or prophylactic agentof a formulae herein to a subject (e.g., a mammal, such as a human).Thus, one embodiment is a method of treating a subject suffering from orsusceptible to an infection, trauma, wound, open fracture, or relateddisease or disorder that requires targeting of a therapeutic compositionto a site. The method includes the step of administering to the mammal atherapeutic amount of a compound herein sufficient to treat the diseaseor disorder or symptom thereof, under conditions such that the diseaseor disorder is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for an infection, in need of healing,having a trauma, wound, open fracture, or related disease, disorder, orsymptom thereof. Determination of those subjects “at risk” can be madeby any objective or subjective determination by a diagnostic test oropinion of a subject or health care provider (e.g., genetic test, enzymeor protein marker, Marker (as defined herein), family history, and thelike). The agents herein may be also used in the treatment of any otherdisorders in which it is desirable to promote healing or treat orprevent an infection.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., wound healing parameters, number ofbacterial cells, or any target delineated herein modulated by a compoundherein, C-reactive protein, cytokine levels, or indicator thereof, etc.)or diagnostic measurement (e.g., screen, assay) in a subject sufferingfrom or susceptible to an infection, disorder or symptoms thereof, inwhich the subject has been administered a therapeutic amount of achitosan composition (e.g., a chitosan composition comprising atherapeutic or prophylactic agent) herein sufficient to treat thedisease or symptoms thereof. The level of Marker determined in themethod can be compared to known levels of Marker in either healthynormal controls or in other afflicted patients to establish thesubject's disease status. In preferred embodiments, a second level ofMarker in the subject is determined at a time point later than thedetermination of the first level, and the two levels are compared tomonitor the course of disease or the efficacy of the therapy. In certainpreferred embodiments, a pre-treatment level of Marker in the subject isdetermined prior to beginning treatment according to this invention;this pre-treatment level of Marker can then be compared to the level ofMarker in the subject after the treatment commences, to determine theefficacy of the treatment.

Test Compounds and Extracts

In general, therapeutic compounds suitable for delivery from a chitosancomposition are known in the art or are identified from large librariesof both natural product or synthetic (or semi-synthetic) extracts orchemical libraries or from polypeptide or nucleic acid libraries,according to methods known in the art. Those skilled in the field ofdrug discovery and development will understand that the precise sourceof test extracts or compounds is not critical to the screeningprocedure(s) of the invention. Compounds used in screens may includeknown compounds (for example, known therapeutics used for other diseasesor disorders). Alternatively, virtually any number of unknown chemicalextracts or compounds can be screened using the methods describedherein. Examples of such extracts or compounds include, but are notlimited to, plant-, fungal-, prokaryotic- or animal-based extracts,fermentation broths, and synthetic compounds, as well as modification ofexisting compounds.

Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofchemical compounds, including, but not limited to, saccharide-, lipid-,peptide-, and nucleic acid-based compounds. Synthetic compound librariesare commercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds tobe used as candidate compounds can be synthesized from readily availablestarting materials using standard synthetic techniques and methodologiesknown to those of ordinary skill in the art. Synthetic chemistrytransformations and protecting group methodologies (protection anddeprotection) useful in synthesizing the compounds identified by themethods described herein are known in the art and include, for example,those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91:11422,1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al.,Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl.33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994;and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired,any library or compound is readily modified using standard chemical,physical, or biochemical methods.

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84,1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S.Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids(Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage(Scott and Smith, Science 249:386-390, 1990; Devlin, Science249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382,1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra.).

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their activity should be employed wheneverpossible.

When a crude extract is identified as containing a compound of interest,further fractionation of the positive lead extract is necessary toisolate chemical constituents responsible for the observed effect. Thus,the goal of the extraction, fractionation, and purification process isthe careful characterization and identification of a chemical entitywithin the crude extract that achieves a desired biological effect.Methods of fractionation and purification of such heterogenous extractsare known in the art.

Small molecules of the invention preferably have a molecular weightbelow 2,000 daltons, more preferably between 300 and 1,000 daltons, andmost preferably between 400 and 700 daltons. It is preferred that thesesmall molecules are organic molecules.

Kits

The invention provides kits that include chitosan compositions. In oneembodiment, the kit includes a chitosan composition containing atherapeutic or prophylactic agent that that prevents or treats infection(e.g., an antimicrobial agent) or that promotes healing (e.g., growthfactor, anti-inflammatory, clot promoting agent, anti-thrombotic). Inother embodiments, the kit contains a therapeutic device, such as achitosan film useful in wound healing, chitosan sponge, hydrogel, orimplant/prosthetic device comprising a chitosan composition describedherein. If desired, the aforementioned chitosan compositions furthercomprise an agent described herein.

In some embodiments, the kit comprises a sterile container whichcontains a chitosan composition; such containers can be boxes, ampoules,bottles, vials, tubes, bags, pouches, blister-packs, or other suitablecontainer forms known in the art. Such containers can be made ofplastic, glass, laminated paper, metal foil, or other materials suitablefor holding medicaments.

If desired a chitosan composition of the invention is provided togetherwith instructions for using it in a prophylactic or therapeutic methoddescribed herein. The instructions will generally include informationabout the use of the composition for the treatment of a trauma,infection or related disease in a subject in need thereof. Theinstructions may be printed directly on the container (when present), oras a label applied to the container, or as a separate sheet, pamphlet,card, or folder supplied in or with the container.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 Antibiotic Delivery by a Chitosan Sponge InhibitedBacterial Growth

There is a need for a biocompatible, resorbable carrier for use incontaminated extremity injuries that can be custom loaded withtherapeutic agents based on the suspected bacterial species in a wound.Non-restrictive loading could potentially reduce bacterial colonizationby orders of magnitude and reduce infection rates and loss offunctionality in limbs in compromised patients with contaminated wounds.The following results indicate that lyophilized chitosan sponges areuseful as a carrier for antibiotics. Such sponges may be used alone oras an adjunctive therapy to standard irrigation and debridement fororthopaedic trauma and other musculoskeletal applications.

A sponge containing 61 and 71% deacetylated (DDA) chitosan was loadedwith the antibiotic amikacin. Amikacin release at one hour was found tobe 88.7±2.4 μg/ml and 83.7±7.3 μg/ml for the 61 and 71 DDA samples,respectively. Amikacin was evident at 72 hours as the 61 DDA samplesreleased 2.8±1.3 μg/ml and the 71 DDA samples released 2.2±1.9 μg/ml(FIG. 1A). Bacterial growth inhibition of P. aeruginosa was found to be98.7% after one hour and 93.1% after 72 hours.

The effect of neutralization conditions on amikacin elution fromchitosan sponges was also analyzed (FIG. 1B). In this test, thefollowing chitosan sponges were evaluated: (1) 61 DDA chitosan, 0.5MNaOH neutralized, (2) 71 DDA chitosan 1 M NaOH neutralized, (3) 61 DDAchitosan 1 M NaOH neutralized, (4) 71 DDA chitosan 2 M NaOH neutralized,(5) 61 DDA 2M NaOH neutralized. All sponges were submerged in 5 mg/mlamikacin solution for 2 minutes prior to elution testing. The elutionwas done in 50 ml of sterile saline solution and completely refreshed ateach time point. The elution was carried out for 7 days. The valuesshown on the graph are in μg/ml. In general, 61DDA chitosan neutralizedwith 0.5M NaOH provided the best long term release

Agent elution from chitosan films was also analyzed (FIG. 1C).Sterilized chitosan films were loaded with amikacin and vancomycin. Allfilms were submerged in 5 mg/ml amikacin or vancomycin solution for 2minutes prior to elution testing. The elution was done in 50 ml ofsterile saline solution and completely refreshed at each time point. Theelution was carried out for 3 days. The values shown in FIG. 1C areexpressed in μg/ml.

The ability to customize the antibiotic choice is advantageous becauseit allows clinicians to tailor treatment regimens based on known orsuspected bacterial species present. Chitosan sponges allow for largeruptake of antibiotic solution and higher release concentrations thanchitosan films. The sponges are also customizable in terms ofdegradation as manufacturing alterations (e.g., degree of deacetylation,neutralization solution, solvent make-up, and chitosan weight %, and/orcrystallinity) can alter degradation rates. The results indicate thatincorporation of antibiotics into chitosan provides a local drugdelivery system that can be used alone or in conjunction with irrigationand debridement therapies.

Example 2 Chitosan Compositions Comprising Antibiotics that InhibitedInfection In Vivo

Chitosan sponges (FIGS. 2A-2C) were loaded with antibiotic. Theantibiotic elution profile is provided at Table 1.

TABLE 1 Results of the amikacin elution study performed with sterilizedchitosan sponges using the finalized methodology in sponge fabrication.Sample/ 1 hr 3 hr 6 hr 24 hr 48 hr 72 hr Time (μg/ml) (μg/ml) (μg/ml)(μg/ml) (μg/ml) (μg/ml) 1 400 400 200 100 100 100 2 400 400 100 200 100100 3 400 400 100 200 100 100 All values listed are in μg/ml. Allsamples were drawn from 20 ml of 1 × PBS (complete refreshment ofsolution was done at each timepoint).

Agent elution from chitosan sponges loaded with the antibioticsamikacin, vancomycin, and daptomycin was analyzed in parallel (FIG. 17).As shown in FIG. 17, profiles for amikacin and vancomycin release wassimilar throughout the duration of the study whereas daptomycin releasewas significantly less than the other two tested antibiotics at the 72hour time point. Agent elution from chitosan sponges loaded with morethan one antibiotic was also analyzed. Amikacin elution from chitosansponges was measured when chitosan sponges were loaded with amikacinalone, amikacin+vancomycin, and amikacin+daptomycin (FIG. 18). As shownin FIG. 18, vancomycin nor daptomycin affected the release of amikacin.Vancomycin elution from chitosan sponges was measured when chitosansponges were loaded with vancomycin alone and vancomycin+amikacin (FIG.19). As shown in FIG. 19, amikacin did not affect the release ofvancomycin. Daptomycin elution from chitosan sponges was measured whenchitosan sponges were loaded with daptomycin alone anddaptomycin+amikacin (FIG. 20). As shown in FIG. 20, amikacin did notaffect the release of vancomycin.

To determine whether agents eluted from the sponges could be used toinhibit bacterial growth, zone of inhibition (ZOI) studies were carriedout (FIGS. 3A-3D). Chitosan sponges loaded with a single antibiotic orcombinations of antibiotics, as above, were analyzed for their abilityto inhibit bacterial growth. For the assays to determine inhibition ofbacterial growth, the bacterial strains Pseudomonas aeruginosa andStaphylococcus aureus were used. Results showing the inhibition ofPseudomonas aeruginosa and Staphylococcus aureus are shown at Tables 2and 3, respectively.

TABLE 2 Chitosan sponges loaded with antibiotics inhibited growth ofPseudomonas aeruginosa P. aeruginosa 1 3 6 24 48 72 amikacin only − − −− + + amikacin + vancomycin − − − − + + amikacin + daptomycin − −− + + + Bacterial growth of Pseudomonas aeruginosa in the presence ofsamples from the elution studies (FIGS. 17-20). In each column, growthin defined with a (+) and lack of growth is defined with a (−). (n = 3)As shown in Table 2, none of the groups tested inhibited bacterialgrowth at 48 or 72 hours. Lack of inhibition was also observed at 24hours with the amikacin+daptomycin group.

TABLE 3 Chitosan sponges loaded with antibiotics inhibited growth ofStaphylococcus aureus S. aureus 1 3 6 24 48 72 amikacin only − − − − − −vancomycin only − − − − − − daptomycin only − − − − − − amikacin +daptomycin − − − − − − amikacin + vancomycin − − − − − + Bacterialgrowth of Staphylococcus aureus in the presence of samples from elutionstudies (FIGS. 17-20). In each column, growth in defined with a (+) andlack of growth is defined with a (−). (n = 3)As shown in Table 3, all groups tested inhibited growth of S. aureusthrough 72 hours except the group containing amikacin+vancomycin, whichshowed lack of inhibition at the 72 hour timepoint.

Elution data from chitosan films is provided at Table 4. Films weresubmerged in 5 mg/ml amikacin or vancomycin solution for 2 minutes priorto elution testing. The elution was done in 50 ml of sterile salinesolution and completely refreshed at each time point. The elution wascarried out for 3 days. The values shown on the graph are in μg/ml.

TABLE 4 Elution data from amikacin and vancomycin loaded f Antibiotic 1hour 3 hours 6 hours 24 hours 48 hours 72 hours Amikacin 50.63 ±  2.15 ±3.17 ± 3.93 ±  4.7 ±  0.6 ± (μg/ml)  4.49 1.13 1.14 0.57 2.49 0.48Vancomycin 83.45 ± 19.35 ± 0.58 ± 2.53 ± 9.72 ± 6.13 ± (μg/ml) 17.257.98 0.88 0.66 1.92 0.39 Table 4 displays amikacin and vancomycinelution from sterilized chitosan films corresponding to FIG. 1C.

Sponges were also used in a well-established, contaminated tibialfracture model in goats. This model mimics an orthopaedic trauma injuryas seen in military combat due to Improvised Explosive Devices (IEDs).This model contains muscle, bone, and tissue injuries with thermaldamage to each. The sponges were hydrated in amikacin (P. aeruginosa) orvancomycin (S. aureus) [5 mg/ml solution] for 60 seconds. The goats wereeuthanized at 48 hours and imaged a third time to quantify bacterialevels (FIG. 5D). Blood was drawn to monitor serum levels of antibioticat 6, 12, 24, 36, and 42 hours. Results are shown in FIG. 6 and in Table5 (below).

The wound was inoculated with Pseudomonas aeruginosa (lux) orStaphylococcus aureus (lux) bacteria and closed for 6 hours. FIGS. 4A-4Dshow quantitation of bacteria present in the wound at the indicated timepoint. After 6 hours, the wound was opened and imaged to obtainbacterial luminescent intensity (FIG. 5A-5D). The amount of lightintensity was recorded as a baseline value for bacterial contamination.The wound was debrided and irrigated with 9 L of sterile salinesolution. After irrigation, the wound was imaged a second time. Goats inthe treatment group received an antibiotic-loaded chitosan sponge, whichwas placed in the wound before the wound was closed.

In the absence of local administration of an antibiotic using a chitosansponge, high levels of bacteria were found in wounds despite irrigationand debridement. Goat 841 had a 65% reduction in bacterial cell countafter irrigation and debridement. This value rebounded to 214% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment. Goat 843 had a 61% reduction in bacterial cell count afterirrigation and debridement. This value rebounded to 91% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment. Goat 845 had a 93% reduction in bacterial cell count afterirrigation and debridement. This value rebounded to 56% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment. Goat 846 had a 95% reduction in bacterial cell count afterirrigation and debridement. This value rebounded to 20% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment. Goat 847 had an 89% reduction in bacterial cell count afterirrigation and debridement. This value rebounded to 30% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment.

In contrast, goats that received local administration of amikacin via achitosan sponge had virtually no Pseudomonas aeruginosa bacteria presentin their wounds. Goat 8 had an 87% reduction in bacterial cell countafter irrigation and debridement. This value was reduced to 0% after 42hour treatment with an amikacin-loaded chitosan sponge. Goat 32 had an91% reduction in bacterial cell count after irrigation and debridement.This value was reduced to 1% after 42 hour treatment with anamikacin-loaded chitosan sponge. Goat 839 had an 89% reduction inbacterial cell count after irrigation and debridement. This value wasreduced to 1% after 42 hour treatment with amikacin-loaded chitosansponge. Goat 842 had an 87% reduction in bacterial cell count afterirrigation and debridement. This value was reduced to 0% after 42 hourtreatment with amikacin-loaded chitosan sponge. Goat 2026 had an 81%reduction in bacterial cell count after irrigation and debridement. Thisvalue was reduced to 0% after 42 hour treatment with amikacin-loadedchitosan sponge.

Similar results were observed when vancomycin-loaded chitosan spongeswere used. In the control group, goats showed high levels of bacteria(Staphylococcus aureus) in their wounds despite irrigation anddebridement. Goat 14069 had a 22% reduction in bacterial cell countafter irrigation and debridement. This value rebounded to 2464% of6-hour pre-irrigation and debridement cell count after 42 hours with noother treatment. Goat 7147 had an 81% reduction in bacterial cell countafter irrigation and debridement. This value rebounded to 159% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment. Goat 20 had a 73% reduction in bacterial cell count afterirrigation and debridement. This value rebounded to 157% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment. Goat 94 had a 76% reduction in bacterial cell count afterirrigation and debridement. This value rebounded to 357% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment. Goat 263 had a 59% reduction in bacterial cell count afterirrigation and debridement. This value rebounded to 249% of 6-hourpre-irrigation and debridement cell count after 42 hours with no othertreatment.

Importantly, goats treated with chitosan sponges loaded with vancomycindid not show the Staph aureus bacterial rebound observed in the controlgroup. For example, Goat 840 had a 74% reduction in bacterial cell countafter irrigation and debridement. This value was reduced to 0% after 42hour treatment with a vancomycin-loaded chitosan sponge. Goat 155 had a72% reduction in bacterial cell count after irrigation and debridement.This value was reduced to 1% after 42 hour treatment with avancomycin-loaded chitosan sponge. Goat 837 had an 88% reduction inbacterial cell count after irrigation and debridement. This value wasreduced to 7% after 42 hour treatment with a vancomycin-loaded chitosansponge.

TABLE 5 Serum levels of amikacin and vancomycin drawn over 42 hours.Amikacin concentrations Vancomycin concentrations Conc Conc Time (mean ±standard deviation) Time (mean ± standard deviation)  6 hr 0.526 ± 0.478(ug/ml)  6 hr 1.630 ± 0.646 (ug/ml) 12 hr 0.200 ± 0.000 (ug/ml) 12 hr0.703 ± 0.348 (ug/ml) 24 hr 0.200 ± 0.000 (ug/ml) 24 hr 0.845 ± 0.539(ug/ml) 36 hr 0.200 ± 0.000 (ug/ml) 36 hr 0.740 ± 0.477 (ug/ml) 42 hr0.200 ± 0.000 (ug/ml) 42 hr 0.275 ± 0.070 (ug/ml) The data given aboveis the actual antibiotic levels found in goat serum during a establishedfracture/wound model. The levels taper off from the initial 6 hour drawto the conclusion of the 42 hour study. All values are given in μg/ml.

Sponge fragments remained in 8 of the 10 goats treated with chitosansponges. The values listed in Table 6 (below) show the antibioticremaining in each fragment, which was eluted by soaking the spongefragment for 12 hours in sterile saline solution. The values werenormalized to the mass of the sponge remnants, hence the values listedare in μg/ml/g. Goats 8, 32, 839, and 842 were treated with amikacin andgoats 155, 837, 840, and 2437 were treated with vancomycin.

TABLE 6 Antibiotic Elution from Remaining Sponge Fragments 12 hr TDxreading Goat #/ 12 hr TDx reading Goat #/timepoint (ug/ml/g) timepoint(ug/ml/g) 8 1.60 155 4.18 32 0.25 837 2.31 839 0.48 840 2.28 842 0.082437  6.36 Mean: 0.60 Mean: 3.78 Std Dev: 0.68 Std Dev: 1.93 Valueslisted are amikacin (8,32,838,842) and vancomycin (155,837,840,2437)concentrations after 12 hours in sterile saline solution. The sampleswere retrieved from a goat after 42 hours in an in vivo study. Valueswere normalized to account for initial mass of the remaining sponge.Samples were submerged in 20 ml of sterile saline solution and kept at37° C. for 12 hours.

At the time the sponge fragments were removed, it appeared that all ofthe implanted sponges had degraded between 60-100% after 42 hourspost-implantation. The sponges were designed to elute and degrade over72 hours. In vitro testing confirmed a 72 hour degradation profile.

These results indicated that chitosan provides a degradable carriermatrix capable of delivering antibiotic agents locally alone or as anadjunctive therapy to standard treatment methods for traumaticorthopaedic injuries. The prevention of infection from orthopaedictrauma and surgical sites is of significant interest to the healthcarecommunity. The use of a chitosan sponge as an early stage adjunctivetreatment has significantly improved bacterial eradication in both thePseudomonas aeruginosa and Staphylococcus aureus models (FIGS. 4A and4B). In vitro analyses of these chitosan sponges has provided valuableinformation with respect to optimal manufacturing to tune degradationand elution rates. In vivo evaluation indicated that chitosan acts as abiocompatible, biodegradable carrier for antibiotics and successfullydelivered these agents to aid in bacterial eradication in complex,contaminated wounds.

Example 3 Chitosan Films Maintain Mechanical Integrity afterRe-Hydration

The following study shows that chitosan films can be used as anadaptable implant for musculoskeletal wound infection prevention ortreatment. Dehydrated chitosan film absorb antibiotics and elute drugsover time. Chitosan films also biodegrade in the body. The resultsprovided below show that a chitosan drug delivery device can becustomized by a clinician through in situ antibiotic loading. Suchcompositions maintain rehydrated mechanical integrity.

During in situ loading the chitosan film absorbs antibiotics throughrehydration. In one embodiment, antibiotic loading is carried out in anoperating room or other clinical setting, immediately prior toimplantation. This allows a clinician to tailor the drug delivery deviceto the patient's need through antibiotic choice and concentration. Theseproperties allow for chitosan's use in infected musculoskeletal woundtreatment. As shown herein, use of the device prevents and/or treatsinfection through localized antibiotic delivery, provides for attachmentof chitosan films to implant devices employing chitosan's adhesionproperties, and chitosan's degradation in the body eliminates the needfor an additional surgery to recover an implanted device.

To customize chitosan film's degradation and drug delivery, the degreeof chitosan deacetylation (DDA) is varied at 61, 71, and 80% while thefilm's acid solvent was varied between acetic (HAc) and lactic (LA)acids. Strength and elasticity measurements were obtained using thedevice shown in FIG. 12. Strength and elasticity results using UTS andYoung's modulus indicated that 71 and 80% degree of deacetylation filmswith acetic acid solvent had the highest strength and elasticity (FIGS.9, 10, and 13). Two antibiotics, daptomycin (D) and vancomycin (V), wereused to treat drug susceptible Staphylococcus aureus (FIGS. 7, 8, 11Aand B, 14A and 14B). Two-way ANOVA was performed to determine if therewas any interaction among DDA and acid solvent independent variables.For antibiotic uptake, swelling ratio, UTS, Young's modulus, andadhesive strength there was no statistical interaction between DDA andacid solvent (p≧0.0674).

Films using 80% degree of deacetylation absorbed large quantities ofantibiotics indicating they are useful as drug delivery devices (FIG.7). The uptake of daptomycin and vancomycin indicated the amount ofantibiotic a dehydrated film could absorb in 1 minute in terms ofmilligrams of antibiotic per grams of chitosan (FIG. 7). As used herein,daptomycin is abbreviated as D, vancomycin as V, and neither as N; thenumbers 61, 71, and 80 are used to indicate the % degree ofdeacetylation; the acid solvents, lactic acid and acetic acid, areabbreviated LA and HAc, respectively. Film variations V80HAc, D61LA,D71LA, and D80LA were all statistically similar (p≦0.7769) and absorbedsignificantly more antibiotic than other variations (p<0.0001). Whenabsorbing daptomycin, lactic acid films had a significantly higherabsorption than acetic acid films (p<0.0001). When absorbing vancomycin,acetic acid films had a significantly higher absorption than lactic acidfilms (p<0.0001). Lactic acid films absorbed daptomycin at an average of270.80±125.08 mg/g which was significantly higher (p<0.0001) than itsaverage exclusion of vancomycin at −23.12±31.67 mg/g. Conversely, aceticacid films absorbed vancomycin at an average of 118.39±101.93 mg/g,which was significantly higher (p<0.0001) than its average exclusion ofdaptomycin at −46.43±77.56 mg/g. Chitosan films absorbed antibioticsdifferently depending on both the type of acid and DDA, with bestresults occurring when lactic acid films having 80% degree ofdeacetylation absorb daptomycin and acetic acid films having 80% degreeof deacetylation absorbs vancomycin. There was an upward trend ofincreasing antibiotic absorption as the film's degree of deacetylationincreased. Antibiotic exclusion from the chitosan film during in situloading was indicated by a negative uptake value.

The swelling ratio indicated the increase in volume of the 1 minuterehydrated films in percentage of volume increased (FIG. 8). After 1minute in PBS alone, results indicated that the films approximatelydoubled in volume. Non-loaded films exhibited an approximate 100%swelling or doubling in film volume. Analysis indicated that D80LA andV80HAc variations' swelling ratios were similar (p=0.8381) andsignificantly higher than every other variation (p<0.0001), except forV71HAc (p=0.1703 and 0.0852 respectively). Generally, when the filmswere in an antibiotic environment that favored increased uptake, thefilms had a higher swelling ratio.

The UTS and the Young's modulus of the dry/dehydrated films are shown inFIGS. 9 and 10. The 71HAc and 80HAc groups exhibited UTS values of55.47±11 and 49.89±3.12 MPa which were statistically similar (p=0.2131).These films also exhibited UTS that were significantly higher than theother test groups (p<0.0001). Similarly, Young's moduli for the 71HAcand 80HAc were statistically similar (810.99±207.64, 762.53±49.62,p=0.06597) and significantly greater than other test groups (p<0.0001).No differences were detected in UTS or elastic modulus values of theother test groups.

Films using lactic acid showed the benefit of increased adherence toimplant alloys. A chitosan film having an 80% degree of deacetylationprovides desirable amounts of antibiotic and mechanical properties thatindicate it is ideally suited for drug delivery to a site of trauma.Adhesion testing indicated the adhesion strength, or the maximum tensileload per area in kPa (FIG. 13). Adhesive strength was measured accordingto standards promulgated by ASTM International (Designation D5179-02,www.astm.org/Standards/D5179.htm, which is incorporated herein byreference in its entirety). Ti71LA, SS61LA, and SS80LA alloy/filmvariations were statistically similar, and apart from those variations,Ti71LA had significantly higher adhesive strength than every otheralloy/film combination (p≦0.0310). Generally, acetic acid filmvariations had lower adhesive strength than lactic acid variations (LA:˜7-10 kPa v. HAc: ˜4-7 kPA). In acetic acid films there was an upwardtrend in adhesive strength as degree of deacetylation increases.Additional studies indicated that in situ antibiotic loading did nothave a statistically significant effect on adhesion strength whencompared to rehydration alone. Film adhesion of chitosan films in situloaded with antibiotics did not significantly differ from that ofnon-loaded chitosan films, suggesting adhesive uses of theantibiotic-loaded chitosan films (e.g., as an adjunctive antibiotic wrapon a musculoskeletal device). In one embodiment, tensile strength is atleast about 45, 50, 55, 60, 65, or 70 mPA. In another embodiment,Young's modulus is at least about 700, 800, 900, or 1000 mPA. In anotherembodiment, the adhesive strength is at least about 6-12 kPA (e.g., 6,7, 8, 9, 10, 11, 12 kPA).

Antibiotic elution results from the films indicated the concentration ofantibiotic present in solution per chitosan film sample weight over aperiod of time given in (mg/ml)/g (FIGS. 11A and 11B). For daptomycin(FIG. 11A), 80LA variations eluted consistently significantly higherquantities of daptomycin over the 72 hour period. This increased elutioncould be correlated to the increased antibiotic uptake (FIG. 7). Invancomycin elution (FIG. 11B), 80HAc variations had a higher averageelution rate. The 72 hour elution's approximate antibiotic release foreach individual film variations remained in the same range, except forV71HAc and D71LA, which showed a short but relatively extended releasein comparison to their maximum eluted concentration, although thisdifference was not significant.

Antibiotic activity was determined using turbidity assays, indicated bythe percent inhibition of S. aureus growth (FIGS. 15A and 15B). Overall,the eluates from the films inhibited S. aureus at all time points. Fourvariations showed variability at their respective time points: V80HAc at12 hrs; V71HAc, D61LA, and D71LA at 24 hrs. However, a potential sourceof variability for V80HAc and V71HAc at 12 and 24 hrs, respectively, mayhave been caused by a precipitous interaction between the eluates and acomponent in the TSB media (FIG. 15B). Ultimately, the 48 and 72 hreluate samples for both vancomycin and daptomycin were found to beactive in inhibiting the growth of S. aureus.

Degradation of chitosan films with and without antibiotics is shown inFIGS. 14A and 14B. The degradation study with lysozyme indicated thechitosan film weight that remained after a period of time in percentageof original film weight (FIGS. 14A and 14B). Film variations with 61%DDA degraded to a lesser extent and with no significant differencebetween antibiotically loaded and non-loaded variations. Variations withhigher DDAs degraded more than those with a lower DDA. When antibioticloading had an effect, the effect was a decrease in the film degradationamount. After approximately 60 hrs, the degradation rate slowedconsiderably as each individual variation's percentages werestatistically similar after that point.

Chitosan films absorb antibiotic differently depending on both the acidused to form the film and the degree of chitosan deacetylation. Ingeneral, optimal results were obtained in films comprising 80LA loadedwith daptomycin and 80HAc loaded with vancomycin. Optimizing thechitosan matrix, antibiotic, and acid solvent produces a film thatprovides for the absorption of multiple antibiotics and that providesfor extended antibiotic release. Advantageously, the film isbiocompatible and can be used in conjunction with prosthetic devices.

Example 4 Antibiotic-Loaded Chitosan Sponges Prevent BacterialColonization on Implanted Catheters in an Established Murine Model

Local drug delivery provides an improved approach to combating theformation of biofilms on implant surfaces. Prophylactic treatment ofimplants with antibiotics to prevent surface colonization and subsequentbiofilm formation reduces the risk of biofilm formation. A chitosanlocal delivery system offers the benefit of bolus antibiotics at theimplant site delivered in a degradable matrix. A chitosan local deliverysystem that is degradable matrix has advantages over non-degradablecarriers such as polymethylmethacrylate (PMMA) beads. Drawbacks to usingPMMA beads include removal surgery, subtherapeutic release ofantibiotics, and bacterial colonization on the implant surface. Thestudy presented below showed that the prophylactic use of a daptomycin-,vancomycin-, or linezolid-loaded chitosan sponge significantly decreasesthe bacterial counts of a CA-MRSA isolate in an established murinecatheter model when compared to PBS-loaded chitosan sponges orcatheter-only controls.

To evaluate the loaded chitosan sponge as a prophylactic treatment inpreventing biofilm formation on an implantable fluorinated ethylenepropylene (FEP) catheter, we used a Staphylococcus aureus strain(FPR3757) that has been described previously. This USA300 strain waschosen due to it having a sequenced genome and the fact that it is aprototype community-acquired methicillin-resistant staphylococcus aureus(CA-MRSA) isolate.

Biofilm formation/inhibition was assessed in vivo using a murine modelof catheter-associated biofilm formation. Five study groups wereevaluated during this study: (a) daptomycin-loaded chitosan sponges, (b)vancomycin-loaded chitosan sponges, (c) linezolid-loaded chitosansponges, (d) phosphate buffered saline (1×PBS)-loaded chitosan sponges,and (e) catheter only. Sterile chitosan sponges (7.0 mm diameter) wereloaded with 125 microliters (μl) of daptomycin solution, vancomycinsolution, linezolid solution, or 1×PBS (groups a-d). Concentrationvalues for daptomycin, vancomycin, and linezolid were 10.0 μg/ml, 20.0μg/ml, and 40.0 μg/ml, respectively. These values correspond to 10 timesthe concentration defined by the Clinical and Laboratory StandardsInstitute (CLSI) as the breakpoint MIC for a drug-sensitive strain of S.aureus. Subcutaneous pockets were created in the hind limb of NIH Swissmice (FIGS. 21A and 21B). Immediately prior to implantation of 1-cmsegments of catheters, chitosan sponges were placed in the wound (groupsa-d) (n=6) (FIGS. 21A and 21B). Because each mouse had two cathetersimplanted, and because preliminary experiments confirmed the absence ofcross-contamination between catheters in opposite flanks of the samemouse, each catheter subsequently was treated as an independent datapoint (n=12). Group (e) received catheter placement with no chitosansponge. After implantation of all catheters (approximately 1 h), 10⁵ CFUof the test strain in a total volume of 100 μl of 1×PBS was introduceddirectly into the lumen of the catheter. Because this study was intendedto evaluate the prophylactic efficacy of the chitosan delivery system toprevent biofilm formation, catheters were harvested after animalsacrifice at 48 h post-inoculation. Catheter segments were harvested andrinsed in sterile PBS to remove non-adherent bacteria. Adherent bacteriawere removed by sonication in 5.0 ml of PBS. Quantification of viablebacteria colonizing each catheter was determined by platingappropriately diluted samples on tryptic soy agar (TSA). Samples wereincubated overnight at 37° C. Quantitative bacterial counts werecalculated based on the number of colonies obtained multiplied by thecorresponding dilution factor.

Bacterial count data was analyzed using non-parametric methods aftertesting for normality. Specifically, data were subjected to theMann-Whitney test followed by Wilcoxon rank-sum pairwise testing. Datawere analyzed using JMP 8 (Cary, N.C.). Data were consideredsignificantly different if p≦0.05.

The efficacy of an antibiotic-loaded, degradable delivery system toprevent the formation of a CA-MRSA biofilm on an implantable cathetersurface in an established murine model was assessed. Groups containingdaptomycin- and vancomycin-loaded chitosan sponges preventedcolonization of bacteria on the implant surface in 100% of the retrievedcatheter segments, 24 of 24 contained no bacteria after plating (FIG.22). The bacterial counts for both groups were 0 CFU/catheter. Groupsthat contained linezolid- and PBS-loaded chitosan sponges as well ascatheter-only controls did not inhibit the formation of MRSA-inducedbiofilms on the FEP catheter surface. The linezolid-loaded chitosansponge group had a bacterial count of 4.19×10⁵ CFU/catheter. ThePBS-loaded chitosan sponge group had a bacterial count of 9.96×10⁵CFU/catheter. The catheter-only group had lower bacterial counts(2.30×10⁵ CFU/catheter) but was not statistically significant.Daptomycin- and vancomycin-loaded chitosan sponge groups differedsignificantly in terms of bacterial counts from the other 3 groups(p<0.0001), but were not statistically different from each other. Groupsc, d, and e were not significantly different from each other.

The results presented in this study indicate that the prophylactic useof an antibiotic-loaded chitosan sponge prevents the colonization andsubsequent biofilm formation on the surface of an implantablebiomaterial. The results suggest that the efficacy of the treatment wasdrug dependant. This finding is similar to those reported in whichdaptomycin was the most efficacious drug used against UAMS-1 (MRSAstrain). However, previous studies differ because the present study alocal antibiotic system is used prophylactically in an in vivo model, asopposed to evaluation of drug activity against MRSA in vitro. In thepresent study, both daptomycin and vancomycin groups were 100%successful in preventing bacterial colonization. The results reported inthis study showed that antibiotic-loaded chitosan sponges have thepotential to be used prophylactically for the prevention of CA-MRSAbiofilm formation.

The results described herein were obtained using the following methodsand materials.

Sponge Preparation

Chitosan sponges were prepared as follows. In one approach, chitosanspongers were prepared by dissolving 4.5 grams (g) of chitosan into295.5 milliliters (ml) of 1% (v/v) acidic solvent (lactic and/or aceticacids). The chitosan was 71% deacetylated (DDA) acquired from Primex(Siglufjordur, Iceland). In another approach, a chitosan solution wasprepared by dissolving 5.0 grams (g) of chitosan into 500 milliters (ml)of 1% (v/v) acidic solvent. The chitosan used was 61 and 71%deacetylated (DDA) from Primex (Iceland). 25 ml of aqueous chitosan wascast into aluminum dishes and frozen for one hour at −80 C. Samples werelyophilized for 48 hours. Sponges were neutralized in sodium hydroxideand washed in distilled water until neutral. Samples were re-frozen andre-lyophilized before sterilization. The sponges were sterilized usinglow-dose gamma irradiation (25-32 kGy).

In a preferred approach adopted for use in the goat wound treatmentmodel described in Example 2, 2.5 grams of chitosan was dissolved in247.5 ml of 1 (v/v) % blended acid solvent. The mixture contained75%/25% lactic to acetic acid. This mixture was stirred for 4-6 hours atmaximum allowable speed on a stir plate. The chitosan solution wasfiltered to remove undissolved chitosan. 25 ml of chitosan solution waspipetted into small aluminum weigh dishes (6 mm diameter). Each dish wasfrozen at −80 C freezer for 1 hour. The frozen samples were then placedinto a freeze-dryer and lyophilize for 48 hours. Sponge samples wereneutralized in 0.175 M NaOH— solution for ˜30-40 seconds. The spongeswere rinsed repeatedly in containers filled with distilled water and pHchanges were monitored until the rinsing water was neutral in pH. There-hydrated sponges were then incubated at −80 C freezer for 1 hour andre-lyophilized for 24-48 hours. The sponges were then sterilized usinglow-dose gamma irradiation at Wright Medical (25-32 kGy).

A composite containing chitosan sponge in chitosan gel (a“sponge-in-gel” composite) can be made from a chitosan gel component anda chitosan sponge component. A gel “matrix” component is prepared bydissolving chitosan (e.g., as described herein), filtering particulate,and allowing the solution to de-gas overnight. Chitosan solution istransferred into a container and frozen for at least 1 hour (−80° C.).The length of time the chitosan solution is frozen can be adapted to thesize of the sponge (e.g., longer freezing time for larger sponges).

After freezing, the frozen samples are lyophilized for ˜48 hours andsterilized via gamma irradiation. The lyophilized sponge is notneutralized and is used as the adhesive “gel” matrix. A “sponge”component is prepared using the lyophilized sponges (e.g., as describedherein). The lyophilized sponges are neutralized by submerging in sodiumhydroxide solution (various concentrations of NaOH may be used).Hydrated sponges are rinsed with water several times before re-freezingfor at least 1 hour (−80° C.). The frozen sponges are lyophilized againfor 36-48 hours. The duration of lyophilization is dependent onlyophilizer and the size of sponge. The “double” lyophilized spongesamples are sterilized via gamma irradiation.

To prepare the“sponge-in-gel” composite, a combination of “gel matrix”and “sponge” components are coarsely ground (e.g., in a standard coffeegrinder). The finer components are the single-lyophilized sponge pieces,and the larger components are the double lyophilized sponge pieces. Inone embodiment, at least about 25%-95% of the composite is hydrogelcomponent. In another embodiment, at least about 5%-75% of the compositeis sponge. The composite is customized based on the adhesivenessrequired and/or the size of the wound. An increased amount ofadhesiveness is desired if the wound is prone to drainage or hasincreased surface area. In another embodiment, an increased amount ofsponge material is desired for a cavity wound. This blended mixture ofsingle- and double-lyophilized chitosan sponge fragments is thenhydrated with a solution (antibiotic, saline, antifungal, etc) to form apaste mixture. The resulting paste has a binding “gel” matrix(single-lyophilized sponge component) with larger, dispersed “sponge”fragments throughout the gel (double-lyophilized sponge component). The“sponge-in-gel” composite can be prepared in a short amount of time. Inone embodiment, the paste is mixed and is delivered at thepoint-of-care. The agent is incorporated at the time the composite ishydrated. In one embodiment, the composite is delivered to a site oftrauma via a sterile syringe.

Advantageously, the composite provides for a complete void fill andprevents migration of the chitosan composition within the wound. Thisfacilitates localized delivery of an agent to the site of trauma. The“gel matrix” typically has greater adherence properties than the spongeportion of the composite. Thus, the amount of “gel matrix” can beincreased or decreased based on the needs of the patient. In oneembodiment, an increased amount of gel matrix (e.g., greater than about50%, 70%, 80%, 90%, 95%) is used to increase tissue adherence. Inanother embodiment, an increase amount of sponge fragments (e.g.,greater than about 50%, 70%, 80%, 90%, 95%) to provide for sustainedelution of an agent over time. Preferably, the composite provides forthe bimodal delivery of an agent. In the first phase, an agent isquickly released from the gel matrix. This first phase of elutiontypically occurs over the course of hours (e.g., 1, 2, 3, 4, 5, 6 or 12hours) or days (e.g., 1, 2, 3 days). The second phase of the biomodalelution involves the sustained release of an agent from the spongeportion of the composite. This phase typically occurs over the course ofdays, or weeks. Desirably, the composite provides for sustained elutionof an agent during the course of the composite's degradation. In oneembodiment, the composite comprises a non-neutralized gel portion. Inanother embodiment, the composite comprises a neutralized spongeportion.

Sponge Elution Tests

Sponges were subjected to elution tests by submerging hydrated spongesinto 20 ml of 1× Phosphate Buffered Saline (PBS), kept in a 37° C.incubator for the duration of the study. Sponges were re-hydrated in 10ml of 5 mg/ml amikacin and vancomycin loaded solution. One ml aliquotswere taken at 1, 3, 6, 24, 48, and 72 hours. Aliquots were tested forantibiotic concentration using a fluorescence polarization immunoassaytechnique (TDx, Abbott Labs, Abbott Park, Ill.).

Antibiotic Activity

Drug activity of the aliquots was tested using a turbidity assay. Twodifferent strains of bacteria were used in this study. Vancomycinsamples were tested against Staphylococcus aureus and amikacin sampleswere tested against both Staphylococcus aureus and Pseudomonasaeruginosa. 200 μl of each aliquot was added to 1.8 ml of Mueller-HintonII broth combined with 20 μl of S. aureus inoculum. Amikacin samples(200 μl) were also added to 1.75 ml of trypticase soy broth (TSB) and 50μl of P. aeruginosa inoculum. Samples were incubated for 24 hours at 37C. Absorbance measurements were recorded after incubation at awavelength of 530 nm (A530).

In other studies, antibiotic activity against S. aureus (Cowan I strain)was determined by utilizing the remaining antibiotic elution samples, intriplicate, in a turbidity assay. In this turbidity study, solutionclarity after sufficient bacterial incubation with antibiotic eluatesindicated bacterial inhibition due to antibiotic activity.

In triplicate, 20 μl of vancomycin and daptomycin eluates wereindividually added to the inoculum containing 1.75 ml of tryptic soybroth (TSB) and 25 μl of S. aureus in 5 ml polystyrene test tubes.Blanks containing neither S. aureus nor eluate samples, positivecontrols containing S. aureus without antibiotic eluates, and negativecontrols containing both S. aureus and high concentration antibioticstandards were mixed and incubated at 37° C. along with the eluatesamples. After 24 hours of incubation, the tubes were vortexed and theabsorbance at 530 nm of each inoculum solution was recorded using aspectrophotometer.

Antibiotic Quantitation

High-pressure liquid chromatography (HPLC) was used to quantify theuptake and elution of the antibiotics vancomycin from MP Biomedicals(Irvine, Calif.) and daptomycin from Cubist Pharmaceuticals (Lexington,Mass.). The Varian (Palo Alto, Calif.) HPLC system comprised a ProStar240 Solvent Delivery, ProStar 410 Autosampler, and ProStar 325 UV-VisDetector modules. Module control and data processing were performedusing Varian's Galaxie Chromatography Data System (v1.8.508.1). BothHPLC separation methods were modified from previous research.

For daptomycin quantification the mobile phase consisted of an HPLCgrade acetonitrile and water (62:38, v/v) solution including 4 mMammonium dihydrogen phosphate brought to a pH of 3.25 using phosphoricacid. Separation was accomplished using a Varian Microsorb-MV C8 column,150 mm length and 4.6 mm inner diameter with a flow rate of 1 ml/min.Daptomycin was detected at 232 nm with a retention time of 13.8 minutes(min). Daptomycin quantification was performed in a temperature range of23.3±1.1° C.

For vancomycin quantification, the mobile phase consisted of a HPLCgrade acetonitrile and water (92:8, v/v) solution including 50 mMammonium dihydrogen phosphate brought to a pH of 4 using phosphoricacid. Separation was accomplished using a Varian Microsorb-MV C₁₈column, 150 mm length and 4.6 mm inner diameter with a flow rate of 1ml/min. Vancomycin was detected at 208 nm with a retention time of 24.4min. Vancomycin quantification was performed in a temperature range of23.3±1.1° C.

Film Elution Tests

In one approach, samples were submerged into 15 ml amikacin solution (5mg/ml) and allowed to hydrate for 2 minutes. Samples were then subjectedto elution tests by submerging the films in 50 ml of 1× PhosphateBuffered Saline (PBS) and agitated in a 37 C incubator for the durationof the study. One ml aliquots were removed at 1, 3, 6, 24, 48, and 72hours. Aliquots were tested for antibiotic concentration using afluorescence polarization immunoassay technique (TDxFLx, Abbott Labs,Abbott Park, Ill.).

In another approach, lactic acid films with three different degrees ofdeacetylation were measured for daptomycin elution, and acetic acidfilms with different degrees of deacetylation were measured forvancomycin elution. The elution experiment was performed in triplicateby submerging films in 50 ml of PBS immediately following in situantibiotic loading at 3 mg/ml of antibiotic. The elution procedureexcluded PBS solution refreshment at each time point. The films werethen incubated at 37° C. and 0.5 ml aliquots were removed at 1, 3, 6,12, 24, 48, and 72 hours. The antibiotic concentrations of eluantsamples were determined using HPLC to obtain an elution profile for eachfilm/antibiotic combination.

Activity Tests

Drug activity of the aliquots was tested using a turbidity assay.Samples were tested against Pseudomonas aeruginosa. Samples (200 μl)were added to 1.75 ml of Trypticase Soy Broth (TSB) and 50 μl of P.aeruginosa inoculum. Samples were incubated for 24 hours at 37 C.Absorbance measurements at 530 nm on a spectrophotometer (BioTek).

Film Preparation

Using three chitosan degree of deacetylations and two acid solvents, sixchitosan variations were evaluated. The numbers 61, 71, and 80 were usedto indicate the % degree of deacetylation (DDA); the acid solvents,lactic acid and acetic acid, are abbreviated LA and HAc, respectively.

Primex ChitoClear (Iceland) chitosan powder at 61, 71, and 80% degree ofdeacetylation with 124, 1480, and 332 mPas viscosities, respectively,was used to create the films. A 1.5% (w/v) chitosan solution wasprepared by dissolving the desired variation in either 1% (v/v) aceticor lactic acid solution, under constant stirring for 24 hours (hr). Toremove insolubilities from the chitosan solution, it was filteredthrough 180 μm nylon, placed in a glass mold and transferred to aconvection oven at 60° C. until dry. The dehydrated film was removed andneutralized by placing it in a NaOH solution followed by rinsing inwater. This neutralized film was allowed to dry at 25° C. In anotherapproach, a chitosan solution that had been filtered through an 180 μmnylon screen was allowed to degas at 20° C. The solution was placed in aflat-bottomed glass dish at approximately 0.8 ml/cm² and the solvent wasallowed to evaporate in a convection oven at 38° C. for 24 hrs. Thisproduced a dried film which was neutralized by dipping the film in 2 Msodium hydroxide for approximately 1 sec, followed by pouring 2 L ofdistilled/deionized water over the film for rinsing. The neutralizedfilms were dried on a large-pore sized nylon screen in a convection ovenat 38° C. for 12 hrs.

In another approach, 2.5 grams of chitosan was dissolved into 247.5 mlof 1 (v/v) % blended acid solvent containing 75%/25% lactic to aceticacid. The mixture was stirred for 4-6 hours at max allowable speed on astir plate. The chitosan solution was filtered to remove undissolvedchitosan, and the filtrate was pipetted into a glass Petri dish, whichwas heated at 37° C. for 18-20 hours. The dried films were removed andneutralized in 2.0 M NaOH⁻ solution for ˜30-40 seconds. The films wererinsed with distilled water and pH changes were monitored until therinsing water was neutral in pH. The re-hydrated films were then frozenat −80° C. freezer for 1 hour and then lyophilized for 24 hours. Thefilms were then sterilized using low-dose gamma irradiation (25-32 kGy).

Film Uptake Studies

An uptake study was performed to determine the quantity of antibioticsolution that each chitosan composition could absorb. Antibiotic uptakedetermines the ability of chitosan film to absorb antibiotics. Thisstudy determined the concentration of both vancomycin and daptomycinthat each chitosan film variation could absorb during 1 minute ofrehydration. a 1 mg/ml vancomycin or daptomycin phosphate-bufferedsaline (PBS) solution was created. Using six replications, films ofknown weights were submerged in 50 ml of the antibiotic solution for 1min, where 1 min is representative of effective operating room usage.The film was then submerged in the antibiotic solution for thirtyseconds and removed. The remaining solution was tested using a highpressure liquid chromatography (Varian, Calif.) method to determine theantibiotic concentration.

This method of antibacterial loading is defined as in situ loading, asopposed to pre-loading, where antibiotics would be incorporated in thechitosan solution during film creation. After 1 minute the film wasremoved and a sample of remaining antibiotic solution was used in HPLCto determine its concentration. Antibiotic uptake was normalized by filmweight and determined using the following relations: Antibioticuptake=[(Initial antibiotic solution concentration−Final antibioticsolution concentration)×Antibiotic solution volume](mg)/(Chitosan filmweight)(mg).

Swelling Ratio.

The swelling ratio of the chitosan films was determined after 1 minsubmergence in the presence and absence of daptomycin and vancomycinsolutions. In order to quantify swelling ratio, the initial volume ofchitosan films were determined using electronic digital calipersaccurate 0.03 mm within a range of 0 to 150 mm. The final volume wasdetermined immediately after the antibiotic uptake procedure wasperformed. This data allowed the swelling ratio after 1 minute to bedetermined using the following relationship: Swelling ratio (%)=(Finalfilm volume−Initial film volume)/(Initial film volume)×100

Absorbed antibiotic quantity was determined using differences inconcentrations. Concurrent with the uptake study, film dimensions weremeasured using digital calipers in order to calculate film volumedifferences, yielding the swelling ratio. Chitosan film swelling ratioquantified the increase in volume as the film rehydrated.

Ultimate Tensile Strength, Young's Modulus

Neutralized films were subjected to tensile testing using a UniversalMaterials Testing Machine (Instron, Norwood, Mass.). Ultimate TensileStrength (UTS) and Young's Modulus determines the strength andelasticity of dry/dehydrated chitosan film variations. Using sixreplications, film variations were punched out into ASTM E8 tensiletesting specimens with an initial 25 mm-gage length and 175 mm² area.Dehydrated film thickness was 0.29±0.6 mm. In some analyses, testspecimens were cut uniformly with gauge lengths and widths of 12.7 mmand 3.5 mm respectively. Using an Instron 33R, model 4465 (Norwood,Mass.) Universal Testing Machine with a 50 N load cell automated byInstron's Bluehill 2 (v2.13) software, the ultimate tensile strength(UTS) and Young's modulus of dehydrated films were determined. Due tothe necessity of controlling the test specimen's precise dimensions, itwas necessary to perform this test using dehydrated chitosan filmsamples. The test specimen was securely placed in the hydraulic gripsand tested in tension at a rate of 1 mm/min with data recorded at 200 msintervals. The testing device software was configured to output UTS,Young's modulus and the breaking point % of elongation.

Adhesive Strength

Adhesive strength measurement investigates the adhesive strength ofwet/rehydrated chitosan films to implant grade alloy fixtures, either316L stainless steel (ASTM F138) or 6-4 titanium (ASTM F136), using amodified ASTM standard (D5179-02). These fixtures were gripped by auniversal testing machine which measured the strength required to pullthe implant alloys apart. All experiments were performed with n≧5.

To determine adhesive strength, six specimen replications from allchitosan film variations were cut into minimal 38×38 mm squares. Filmswere then submerged in 50 ml of PBS solution for 1 min, in order tosimulate the in situ loading procedure, and were then positioned betweencylindrical fixtures with a diameter of 35.1 mm to facilitate theadhesion test. The adhesion testing was modeled from ASTM D5179-02 inorder to be performed in-house. Both Instron Universal Testing Machinehydraulic grips were made to hold either 316L SS (ASTM F138) or Ti(Ti-6A1-4V, ASTM 136) alloy fixtures (FIG. 12). The mechanical fixturesurfaces which faced each other were smoothed by superfinishing to aroughness, R_(a), value of 0.025 μm. The superfinishing on the testingcylinders were used to provide comparison testing, not to replicatetypical implant surfaces. The rehydrated chitosan film was sandwichedbetween the two mechanical fixtures with an automatic compressionpre-load of 15 N. Immediately after reaching the pre-load force, themovable crossheads were reversed at 50 mm/min with data recorded at 30millisecond intervals. Film thickness varied at 0.18±0.8 mm and thesoftware gave data output in maximal force (N) which was converted intoadhesive strength (kPa). Tukey's HSD statistical analysis was performedwith α=0.05 to determine statistical differences between filmvariations.

Chitosan Degradation.

A modified procedure (Tomihata and Ikada, 1997) was used to quantify theantibiotic effect on chitosan degradation. In situ loaded and non-loadedchitosan films groups—the same groups used in the antibiotic elution andactivity experiments with additional non-loaded groups yielded a totalof 12 experimental groups to be tested with five replicates each—weresubjected to degradation testing. The weight of clean 90 mm diameterPetri dishes and dehydrated chitosan films was established. Films markedfor in situ loading were submerged in 50 ml of a standard antibioticsolution and then all films were submerged in 25 ml of 100 μg/ml 2×crystallized, chicken egg white lysozyme (MP Biomedicals) PBS solution.The samples were incubated for 20 hours at 37° C. in a convectionincubator. After the incubation period, the lysozyme solution wasremoved and the films were dehydrated using the same method with theconvection oven. The lysozyme/PBS solution was replaced and film weightswere measured every 20 hours for a total of 100 hours. The new filmweights were measured, which enabled degradation to be expressed as thepercent of the film that remained. The percent of the film that remainedwas determined using the following relationship: Percent Remaining(%)=(Petri dish and film weight at x hours−Petri dish weight)(mg)/(Petridish and initial film weight−Petri dish weight)(mg)×100.

Statistical Analysis

Data is reported as the mean±standard deviation. One-way ANOVA was usedto analyze for statistically significant differences. If statisticallysignificant differences were found, then each pair of variations werecompared using the Student t-test. Differences between chitosan filmvariations were determined using the Student t-test. Two-way ANOVA wasused to identify differences between DDA and acid solvent independentvariables. Analysis was performed using JMP 7.0.1 (Cary, N. Dak.).Statistical significance occurred when p<0.05 and are indicated byeither * or †.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method for producing a biodegradable chitosancomposition having a desired biodegradation profile, the methodcomprising (a) dissolving chitosan having a degree of deacetylation ofat least about 51% in one or more acids in a solvent, wherein the acidand solvent are selected to produce a biodegradable chitosan; (b)forming the chitosan into a desired shape and lyophilizing to reduce thewater content by about 80% 100%; (c) neutralizing the chitosancomposition by contacting the composition with a basic solution, whereinthe basic solution is selected to modulate a physical-mechanicalproperty of the chitosan; (d) washing the chitosan composition withwater until neutral; and (e) lyophilizing the composition of step (c) toreduce the water content by about 80%-100%, thereby producing abiodegradable chitosan composition.
 2. The method of claim 1, furthercomprising incorporating at least one antibacterial or antifungal agentinto the chitosan composition at a point of care.
 3. The method of claim1, wherein the chitosan degree of deacetylation, weight percent,neutralization solution, solvent make-up, and/or crystallinity isselected to customize the biodegradation profile, elution profile,and/or a physical-mechanical property of the chitosan composition.
 4. Amethod for producing a biodegradable chitosan composition comprising anantibacterial or antifungal agent selected by a clinician at a point ofcare, the method comprising (a) dissolving chitosan having a degree ofdeacetylation of at least about 51% in one or more acids in a solvent,wherein the acid and solvent are selected to produce a chitosan thatbiodegrades over at least about one, two, three, four or five days invivo; and (b) forming the chitosan into a desired shape and lyophilizingto reduce the water content by about 80%-100%; (c) neutralizing thechitosan composition by contacting the composition with a basicsolution, wherein the basic solution is selected to modulate aphysical-mechanical property of the chitosan; (d) washing the chitosancomposition with water until neutral; and (e) lyophilizing thecomposition of step (c) to reduce the water content by about 80%-100%;(f) selecting an antibacterial or antifungal agent; and (gincorporatingan effective amount of the agent into the composition at a point ofcare.
 5. The method of claim 1, wherein the method is ex vivo.
 6. Themethod of claim 1, wherein the physical-mechanical property is selectedfrom the group consisting of tensile strength, Young's modulus,swelling, degradation, and a combination thereof.
 7. The method of claim1, wherein the chitosan composition is a wound management device.
 8. Themethod of claim 4, wherein the antibacterial or antifungal agent isselected at the point of care.
 9. The method of claim 1, wherein thedesired shape is obtained by pouring the chitosan into a thin layer andheating the chitosan to form a dehydrated chitosan film.
 10. The methodof claim 1, wherein the chitosan is treated with an acid selected fromthe group consisting of acetic, citric, oxalic, proprionic, ascorbic,hydrochloric, formic, salicylic and lactic acids.
 11. The method ofclaim 10, wherein the acid solvent comprises lactic acid and/or aceticacid.
 12. A chitosan composition produced by the method of claim
 1. 13.A wound management device comprising a chitosan composition produced bythe method of claim
 1. 14. A method for treating a bacterial or fungalinfection in a subject at a site of trauma, the method comprisingcontacting the site with a wound management device of claim 13 and aneffective amount of at least one anti-bacterial or anti-fungal agentselected at a point of care.
 15. A method for the local delivery of anantibacterial or antifungal agent to a site of a subject, the methodcomprising contacting the site with a chitosan composition according tothe method of claim 1 comprising an antibacterial or antifungal agentselected at the point of care, thereby delivering the agent to the site.16. A medical device for implantation and delivery of an antibacterialor antifungal agent to the implantation site comprising a chitosancomposition made according to the method of claim
 1. 17. A kitcomprising a chitosan composition of claim 1.